Rationally-designed single-chain meganucleases with non-palindromic recognition sequences

ABSTRACT

Disclosed are rationally-designed, non-naturally-occurring meganucleases in which a pair of enzyme subunits having specificity for different recognition sequence half-sites are joined into a single polypeptide to form a functional heterodimer with a non-palindromic recognition sequence. The invention also relates to methods of producing such meganucleases, and methods of producing recombinant nucleic acids and organisms using such meganucleases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/858,986, filed Sep. 18, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/723,840, filed May 28, 2015, which is acontinuation of U.S. patent application Ser. No. 13/897,923, filed May20, 2013, which is a continuation of U.S. patent application Ser. No.12/771,163, filed Apr. 30, 2010, now U.S. Pat. No. 8,445,251, which is acontinuation of International Patent Application PCT/US2008/082072,filed Oct. 31, 2008, which claims priority to U.S. ProvisionalApplication No. 61/001,247 filed Oct. 31, 2007, the entire disclosuresof which are incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 20, 2012, isnamed 2000706-00125US7.txt and is 57,853 bytes in size.

FIELD OF THE INVENTION

The invention relates to the field of molecular biology and recombinantnucleic acid technology. In particular, the invention relates torationally-designed, non-naturally-occurring meganucleases in which apair of enzyme subunits having specificity for different recognitionsequence half-sites are joined into a single polypeptide to form afunctional heterodimer with a non-palindromic recognition sequence. Theinvention also relates to methods of producing such meganucleases, andmethods of producing recombinant nucleic acids and organisms using suchmeganucleases.

BACKGROUND OF THE INVENTION

Genome engineering requires the ability to insert, delete, substituteand otherwise manipulate specific genetic sequences within a genome, andhas numerous therapeutic and biotechnological applications. Thedevelopment of effective means for genome modification remains a majorgoal in gene therapy, agrotechnology, and synthetic biology (Porteus etal. (2005), Nat. Biotechnol. 23: 967-73; Tzfira et al. (2005), TrendsBiotechnol. 23: 567-9; McDaniel et al. (2005), Curr. Opin. Biotechnol.16: 476-83). A common method for inserting or modifying a DNA sequenceinvolves introducing a transgenic DNA sequence flanked by sequenceshomologous to the genomic target and selecting or screening for asuccessful homologous recombination event. Recombination with thetransgenic DNA occurs rarely but can be stimulated by a double-strandedbreak in the genomic DNA at the target site. Numerous methods have beenemployed to create DNA double-stranded breaks, including irradiation andchemical treatments. Although these methods efficiently stimulaterecombination, the double-stranded breaks are randomly dispersed in thegenome, which can be highly mutagenic and toxic. At present, theinability to target gene modifications to unique sites within achromosomal background is a major impediment to successful genomeengineering.

One approach to achieving this goal is stimulating homologousrecombination at a double-stranded break in a target locus using anuclease with specificity for a sequence that is sufficiently large tobe present at only a single site within the genome (see, e.g., Porteuset al. (2005), Nat. Biotechnol. 23: 967-73). The effectiveness of thisstrategy has been demonstrated in a variety of organisms using chimericfusions between an engineered zinc finger DNA-binding domain and thenon-specific nuclease domain of the FokI restriction enzyme (Porteus(2006), Mol. Ther. 13: 438-46; Wright et al. (2005). Plant J. 44:693-705; Umov et al. (2005). Nature 435: 646-51). Although theseartificial zinc finger nucleases stimulate site-specific recombination,they retain residual non-specific cleavage activity resulting fromunder-regulation of the nuclease domain and frequently cleave atunintended sites (Smith et al. (2000). Nucleic Acids Res. 28: 3361-9).Such unintended cleavage can cause mutations and toxicity in the treatedorganism (Porteus et al. (2005), Nat. Biotechnol. 23: 967-73).

A group of naturally-occurring nucleases which recognize 15-40 base-paircleavage sites commonly found in the genomes of plants and fungi mayprovide a less toxic genome engineering alternative. Such“meganucleases” or “homing endonucleases” are frequently associated withparasitic DNA elements, such as group I self-splicing introns andinteins. They naturally promote homologous recombination or geneinsertion at specific locations in the host genome by producing adouble-stranded break in the chromosome, which recruits the cellularDNA-repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95).Meganucleases are commonly grouped into four families: the LAGLIDADG(SEQ ID NO: 55) family, the GIY-YIG family, the His-Cys box family andthe HNH family. These families are characterized by structural motifs,which affect catalytic activity and recognition sequence. For instance,members of the LAGLIDADG (SEQ ID NO: 55) family are characterized byhaving either one or two copies of the conserved LAGLIDADG (SEQ ID NO:55) motif (see Chevalier et al. (2001), Nucleic Acids Res. 29(18):3757-3774). The LAGLIDADG (SEQ ID NO: 55) meganucleases with a singlecopy of the LAGLIDADG (SEQ ID NO: 55) motif (“mono-LAGLIDADG (SEQ ID NO:55) meganucleases”) form homodimers, whereas members with two copies ofthe LAGLIDADG (SEQ ID NO: 55) motif (“di-LAGLIDADG (SEQ ID NO: 55)meganucleases”) are found as monomers. Mono-LAGLIDADG (SEQ ID NO: 55)meganucleases such as I-CreI, I-CeuI, and I-MsoI recognize and cleaveDNA sites that are palindromic or pseudo-palindromic, while di-LAGLIDADG(SEQ ID NO: 55) meganucleases such as I-SceI, I-AniI, and I-DmoIgenerally recognize DNA sites that are non-palindromic (Stoddard (2006),Q. Rev. Biophys. 38: 49-95).

Natural meganucleases from the LAGLIDADG (SEQ ID NO: 55) family havebeen used to effectively promote site-specific genome modification inplants, yeast, Drosophila, mammalian cells and mice, but this approachhas been limited to the modification of either homologous genes thatconserve the meganuclease recognition sequence (Monnat et al. (1999),Biochem. Biophys. Res. Commun. 255: 88-93) or to pre-engineered genomesinto which a recognition sequence has been introduced (Rouet et al.(1994), Mol. Cell. Biol. 14: 8096-106; Chilton et al. (2003), PlantPhysiol. 133: 956-65; Puchta et al. (1996), Proc. Natl. Acad. Sci. USA93: 5055-60; Rong et al. (2002), Genes Dev. 16: 1568-81; Gouble et al.(2006), J. Gene Med. 8(5):616-622).

Systematic implementation of nuclease-stimulated gene modificationrequires the use of genetically engineered enzymes with customizedspecificities to target DNA breaks to existing sites in a genome and,therefore, there has been great interest in adapting meganucleases topromote gene modifications at medically or biotechnologically relevantsites (Porteus et al. (2005), Nat. Biotechnol. 23: 967-73; Sussman etal. (2004), J. Mol. Biol. 342: 31-41; Epinat et al. (2003), NucleicAcids Res. 31: 2952-62).

I-CreI is a member of the LAGLIDADG (SEQ ID NO: 55) family whichrecognizes and cuts a 22 base-pair recognition sequence in thechloroplast chromosome, and which presents an attractive target formeganuclease redesign. The wild-type enzyme is a homodimer in which eachmonomer makes direct contacts with 9 base pairs in the full-lengthrecognition sequence. Genetic selection techniques have been used tomodify the wild-type I-CreI cleavage site preference (Sussman et al.(2004), J. Mol. Biol. 342: 31-41; Chames et al. (2005). Nucleic AcidsRes. 33: e178: Seligman et al. (2002), Nucleic Acids Res. 30: 3870-9,Amould et al. (2006), J. Mol. Biol. 355: 443-58, Rosen et al. (2006).Nucleic Acids Res. 34: 4791-4800, Amould et al. (2007). J. Mol. Biol.371: 49-65. WO 2008/010009, WO 2007/093918, WO 2007/093836, WO2006/097784, WO 2008/059317, WO 2008/059382, WO 2008/102198, WO2007/060495, WO 2007/049156, WO 2006/097853. WO 2004/067736). Morerecently, a method of rationally-designing mono-LAGLIDADG (SEQ ID NO:55) meganucleases was described which is capable of comprehensivelyredesigning I-CreI and other such meganucleases to targetwidely-divergent DNA sites, including sites in mammalian, yeast, plant,bacterial, and viral genomes (WO 2007/047859).

A major limitation of using mono-LAGLIDADG (SEQ ID NO: 55) meganucleasessuch as I-CreI for most genetic engineering applications is the factthat these enzymes naturally target palindromic DNA recognition sites.Such lengthy (10-40 bp) palindromic DNA sites are rare in nature and areunlikely to occur by chance in a DNA site of interest. In order totarget a non-palindromic DNA site with a mono-LAGLIDADG (SEQ ID NO: 55)meganuclease, one can produce a pair of monomers which recognize the twodifferent half-sites and which heterodimerize to form a meganucleasethat cleaves the desired non-palindromic site. Heterodimerization can beachieved either by co-expressing a pair of meganuclease monomers in ahost cell or by mixing a pair of purified homodimeric meganucleases invitro and allowing the subunits to re-associate into heterodimers (Smithet al. (2006). Nuc. Acids Res. 34:149-157; Chames et al. (2005). NucleicAcids Res. 33:178-186; WO 2007/047859, WO 2006/097854, WO 2007/057781,WO 2007/049095. WO 2007/034262). Both approaches suffer from two primarylimitations: (1) they require the expression of two meganuclease genesto produce the desired heterodimeric species (which complicates genedelivery and in vivo applications) and (2) the result is a mixture ofapproximately 25% the first homodimer, 50% the heterodimer, and 25% thesecond homodimer, whereas only the heterodimer is desired. This latterlimitation can be overcome to a large extent by genetically engineeringthe dimerization interfaces of the two meganucleases to promoteheterodimerization over homodimerization as described in WO 2007/047859,WO 2008/093249, WO 2008/093152, and Fajardo-Sanchez et al. (2008).Nucleic Acids Res. 36:2163-2173. Even so, two meganuclease genes must beexpressed and homodimerization is not entirely prevented.

An alternative approach to the formation of meganucleases withnon-palindromic recognition sites derived from one or moremono-LAGLIDADG (SEQ ID NO: 55) meganucleases is the production of asingle polypeptide which comprises a fusion of the LAGLIDADG (SEQ ID NO:55) subunits derived from two meganucleases. Two general methods can beapplied to produce such a meganuclease.

In the first method, one of the two LAGLIDADG (SEQ ID NO: 55) subunitsof a di-LAGLIDADG (SEQ ID NO: 55) meganuclease can be replaced by aLAGLIDADG (SEQ ID NO: 55) subunit from a mono-LAGLIDADG (SEQ ID NO: 55)meganuclease. This approach was demonstrated by replacing the C-terminalsubunit of the di-LAGLIDADG (SEQ ID NO: 55) I-DmoI meganuclease with anI-CreI subunit (Epinat et al. (2003), Nucleic Acids Res. 31: 2952-62;Chevalier et al. (2002), Mol. Cell 10:895-905; WO 2003/078619). Theresult was a hybrid I-DmoI/I-CreI meganuclease which recognized andcleaved a hybrid DNA site.

In the second method, a pair of mono-LAGLIDADG (SEQ ID NO: 55) subunitscan be joined by a peptide linker to create a “single-chain heterodimermeganuclease.” One attempt to produce such a single-chain derivative ofI-CreI has been reported (Epinat et al. (2003), Nucleic Acids Res. 31:2952-62; WO 2003/078619). However, as discussed herein as well as inFajardo-Sanchez et al. (2008), Nucleic Acids Res. 36:2163-2173, there isnow evidence suggesting that this method did not produce a single-chainheterodimer meganuclease in which the covalently joined I-CreI subunitsfunctioned together to recognize and cleave a non-palindromicrecognition site.

Therefore, there remains a need in the art for methods for theproduction of single-chain heterodimer meganucleases derived frommono-LAGLIDADG (SEQ ID NO: 55) enzymes such as I-CreI to recognize andcut non-palindromic DNA sites.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the development of fusionproteins in which a peptide linker covalently joins two heterologousLAGLIDADG (SEQ ID NO: 55) meganuclease subunits to form a “single-chainheterodimer meganuclease” or “single-chain meganuclease”, in which atleast the N-terminal subunit is derived from a mono-LAGLIDADG (SEQ IDNO: 55) meganuclease, and in which the subunits function together topreferentially bind to and cleave a non-palindromic DNA recognition sitewhich is a hybrid of the recognition half-sites of the two subunits. Inparticular, the invention can be used to genetically engineersingle-chain meganucleases which recognize non-palindromic DNA sequencesthat naturally-occurring meganucleases do not recognize. The inventionalso provides methods that use such meganucleases to produce recombinantnucleic acids and organisms by utilizing the meganucleases to causerecombination of a desired genetic sequence at a limited number of lociwithin the genome of the organism for, inter alia, genetic engineering,gene therapy, treatment of pathogenic infections, and in vitroapplications in diagnostics and research.

Thus, in some embodiments, the invention provides recombinantsingle-chain meganucleases comprising a pair of covalently joinedLAGLIDADG (SEQ ID NO: 55) subunits derived from one or moremono-LAGLIDADG (SEQ ID NO: 55) meganucleases which function together torecognize and cleave a non-palindromic recognition site. In someembodiments, the mono-LAGLIDADG (SEQ ID NO: 55) subunit is derived froma wild-type meganuclease selected from I-CreI, I-MsoI and I-CeuI.

In other embodiments, the invention provides recombinant single-chainmeganucleases comprising a pair of mono-LAGLIDADG (SEQ ID NO: 55)subunits in which the N-terminal subunit is derived from a wild-typemeganuclease selected from I-CreI, I-MsoI and I-CeuI, and the C-terminalsubunit is also derived from a wild-type meganuclease selected fromI-CreI. I-MsoI and I-CeuI, but the N-terminal subunit is derived from awild-type meganuclease of a different species than the C-terminalsubunit.

In some embodiments, the invention provides recombinant single-chainmeganucleases comprising a pair of LAGLIDADG (SEQ ID NO: 55) subunits inwhich the N-terminal subunit is derived from a wild-type meganucleaseselected from I-CreI, I-MsoI and I-CeuI, and the C-terminal subunit isderived from a single LAGLIDADG (SEQ ID NO: 55) subunit from a wild-typedi-LAGLIDADG (SEQ ID NO: 55) meganuclease selected from I-DmoI, I-SceIand I-AniI.

Wild-type mono-LAGLIDADG (SEQ ID NO: 55) meganucleases include, withoutlimitation, the I-CreI meganuclease of SEQ ID NO: 1, the I-MsoImeganuclease of SEQ ID NO: 2, and the I-CeuI meganuclease of SEQ ID NO:3. Wild-type di-LAGLIDADG (SEQ ID NO: 55) meganucleases include, withoutlimitation, the I-DmoI meganuclease of SEQ ID NO: 4, the I-SceImeganuclease of SEQ ID NO: 5, and the I-AniI meganuclease of SEQ ID NO:6.

Wild-type LAGLIDADG (SEQ ID NO: 55) domains include, without limitation,residues 9-151 of the wild-type I-CreI meganuclease of SEQ ID NO: 1;residues 11-162 of the wild-type I-MsoI meganuclease of SEQ ID NO: 2;and residues 55-210 of the wild-type I-CeuI meganuclease of SEQ ID NO:3, residues 9-96 of the wild-type I-DmoI of SEQ ID NO: 4; residues105-178 of the wild-type I-DmoI of SEQ ID NO: 4; residues 32-123 of thewild-type I-SceI of SEQ ID NO: 5; residues 134-225 of the wild-typeI-SceI of SEQ ID NO: 5; residues 4-121 of the wild-type I-AniI of SEQ IDNO: 6; and residues 136-254 of the wild-type I-AniI of SEQ ID NO: 6.

LAGLIDADG (SEQ ID NO: 55) subunits derived from a wild-type LAGLIDADG(SEQ ID NO: 55) meganuclease include, without limitation, subunitsincluding a LAGLIDADG (SEQ ID NO: 55) domain that has at least 85%sequence identity, or 85%-100% sequence identity, to any one of residues9-151 of the wild-type I-CreI meganuclease of SEQ ID NO: 1; residues11-162 of the wild-type I-MsoI meganuclease of SEQ ID NO: 2; andresidues 55-210 of the wild-type I-CeuI meganuclease of SEQ ID NO: 3,residues 9-96 of the wild-type I-DmoI of SEQ ID NO: 4; residues 105-178of the wild-type I-DmoI of SEQ ID NO: 4; residues 32-123 of thewild-type I-SceI of SEQ ID NO: 5; residues 134-225 of the wild-typeI-SceI of SEQ ID NO: 5; residues 4-121 of the wild-type I-AniI of SEQ IDNO: 6; and residues 136-254 of the wild-type I-AniI of SEQ ID NO: 6.

LAGLIDADG (SEQ ID NO: 55) subunits derived from a wild-type LAGLIDADG(SEQ ID NO: 55) meganuclease also include, without limitation, subunitscomprising any of the foregoing polypeptide sequences in which one ormore amino acid modifications have been included according to themethods of rationally-designing LAGLIDADG (SEQ ID NO: 55) meganucleasesdisclosed in WO 2007/047859, as well as other non-naturally-occurringmeganuclease variants known in the art.

In certain embodiments, the invention provides recombinant single-chainmeganucleases comprising a pair of LAGLIDADG (SEQ ID NO: 55) subunitsderived from naturally-occurring LAGLIDADG (SEQ ID NO: 55) subunits eachof which recognizes a wild-type DNA half-site selected from SEQ ID NOs:7-30.

In other embodiments, the invention provides recombinant single-chainmeganucleases comprising a pair of LAGLIDADG (SEQ ID NO: 55) subunitsgenetically engineered with respect to DNA-binding specificity, each ofwhich recognizes a DNA half-site that differs by at least one base froma wild-type DNA half-site selected from SEQ ID NOs: 7-30.

In other embodiments, the invention provides recombinant single-chainmeganucleases comprising a pair of LAGLIDADG (SEQ ID NO: 55) subunits inwhich one subunit is natural and recognizes a wild-type DNA half-siteselected SEQ ID NOs: 7-30 and the other is genetically engineered withrespect to DNA-binding specificity and recognizes a DNA site thatdiffers by at least one base from a wild-type DNA half-site selectedfrom SEQ ID NOs: 7-30.

In some embodiments, the polypeptide linker joining the LAGLIDADG (SEQID NO: 55) subunits is a flexible linker. In particular embodiments, thelinker can include 15-40 residues, 25-31 residues, or any number withinthose ranges. In other particular embodiments, at least 50%, or50%-100%, of the residues forming the linker are polar unchargedresidues.

In other embodiments, the polypeptide linker joining the LAGLIDADG (SEQID NO: 55) subunits has a stable secondary structure. In particularembodiments, the stable secondary structure comprises at least twoα-helix structures. In other particular embodiments, the stablesecondary structure comprises from N-terminus to C-terminus a firstloop, a first α-helix, a first turn, a second α-helix, and a secondloop. In some particular embodiments, the linker can include 23-56residues, or any number within that range.

In another aspect, the invention provides for various methods of use forthe single-chain meganucleases described and enabled herein. Thesemethods include producing genetically-modified cells and organisms,treating diseases by gene therapy, treating pathogen infections, andusing the recombinant single-chain meganucleases for in vitroapplications for diagnostics and research.

Thus, in one aspect, the invention provides methods for producing agenetically-modified eukaryotic cell including an exogenous sequence ofinterest inserted in a chromosome, by transfecting the cell with (i) afirst nucleic acid sequence encoding a meganuclease of the invention,and (ii) a second nucleic acid sequence including said sequence ofinterest, wherein the meganuclease produces a cleavage site in thechromosome and the sequence of interest is inserted into the chromosomeat the cleavage site either by homologous recombination ornon-homologous end-joining.

Alternatively, in another aspect, the invention provides methods forproducing a genetically-modified eukaryotic cell including an exogenoussequence of interest inserted in a chromosome, by introducing ameganuclease protein of the invention into the cell, and transfectingthe cell with a nucleic acid including the sequence of interest, whereinthe meganuclease produces a cleavage site in the chromosome and thesequence of interest is inserted into the chromosome at the cleavagesite either by homologous recombination or non-homologous end-joining.

In another aspect, the invention provides methods for producing agenetically-modified eukaryotic cell by disrupting a target sequence ina chromosome, by transfecting the cell with a nucleic acid encoding ameganuclease of the invention, wherein the meganuclease produces acleavage site in the chromosome and the target sequence is disrupted bynon-homologous end-joining at the cleavage site.

In another aspect, the invention provides methods of producing agenetically-modified organism by producing a genetically-modifiedeukaryotic cell according to the methods described above, and growingthe genetically-modified eukaryotic cell to produce thegenetically-modified organism. In these embodiments, the eukaryotic cellcan be selected from a gamete, a zygote, a blastocyst cell, an embryonicstem cell, and a protoplast cell.

In another aspect, the invention provides methods for treating a diseaseby gene therapy in a eukaryote, by transfecting at least one cell of theeukaryote with one or more nucleic acids including (i) a first nucleicacid sequence encoding a meganuclease of the invention, and (ii) asecond nucleic acid sequence including a sequence of interest, whereinthe meganuclease produces a cleavage site in the chromosome and thesequence of interest is inserted into the chromosome by homologousrecombination or non-homologous end-joining, and insertion of thesequence of interest provides gene therapy for the disease.

Alternatively, in another aspect, the invention provides methods fortreating a disease by gene therapy in a eukaryote, by introducing ameganuclease protein of the invention into at least one cell of theeukaryote, and transfecting the cell with a nucleic acid including asequence of interest, wherein the meganuclease produces a cleavage sitein the chromosome and the sequence of interest is inserted into thechromosome at the cleavage site by homologous recombination ornon-homologous end-joining, and insertion of the sequence of interestprovides gene therapy for the disease.

In another aspect, the invention provides methods for treating a diseaseby gene therapy in a eukaryote by disrupting a target sequence in achromosome of the eukaryotic, by transfecting at least one cell of theeukaryote with a nucleic acid encoding a meganuclease of the invention,wherein the meganuclease produces a cleavage site in the chromosome andthe target sequence is disrupted by non-homologous end-joining at thecleavage site, wherein disruption of the target sequence provides thegene therapy for the disease.

In another aspect, the invention provides methods for treating a viralor prokaryotic pathogen infection in a eukaryotic host by disrupting atarget sequence in a genome of the pathogen, by transfecting at leastone infected cell of the host with a nucleic acid encoding ameganuclease of the invention, wherein the meganuclease produces acleavage site in the genome and the target sequence is disrupted byeither (1) non-homologous end-joining at the cleavage site or (2) byhomologous recombination with a second nucleic acid, and whereindisruption of the target sequence provides treatment for the infection.

These and other aspects and embodiments of the invention will beapparent to one of ordinary skill in the art based upon the followingdetailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a diagram of the structural components of one embodiment of alinker of the invention (Linker 9) and N-terminal and C-terminalresidues of the endonuclease subunits joined by the linker (SEQ ID NO:109).

DETAILED DESCRIPTION OF THE INVENTION 1.1 Introduction

The present invention is based, in part, upon the development of fusionproteins in which a peptide linker covalently joins two heterologousLAGLIDADG (SEQ ID NO: 55) meganuclease subunits to form a “single-chainheterodimer meganuclease” in which the subunits function together topreferentially bind to and cleave a non-palindromic DNA recognition sitewhich is a hybrid of the recognition half-sites of the two subunits. Inparticular, the invention can be used to genetically engineersingle-chain meganucleases which recognize non-palindromic DNA sequencesthat naturally-occurring meganucleases do not recognize.

This discovery has been used, as is described in detail below, to joinmono-LAGLIDADG (SEQ ID NO: 55) meganucleases, which naturally functionas homodimers, into single-chain meganucleases. Further, the discoveryhas been used to join mono-LAGLIDADG (SEQ ID NO: 55) meganucleases whichhave been re-engineered with respect to DNA-recognition specificity intosingle-chain heterodimers which recognize and cleave DNA sequences thatare a hybrid of the palindromic sites recognized by the two meganucleasehomodimers. The invention provides exemplary peptide linker sequencesfor joining LAGLIDADG (SEQ ID NO: 55) subunits into single polypeptides.Importantly, the invention provides a general method for the productionof linker sequences and the selection of fusion points for linkingdifferent LAGLIDADG (SEQ ID NO: 55) subunits to produce functionalrationally-designed single-chain meganucleases.

The invention also provides methods that use such meganucleases toproduce recombinant nucleic acids, cells and organisms by utilizing themeganucleases to cause recombination of a desired genetic sequence at alimited number of loci within the genome of the organism for, interalia, genetic engineering, gene therapy, treatment of pathogenicinfections and cancer, and in vitro applications in diagnostics andresearch.

As a general matter, the invention provides methods for generatingsingle-chain meganucleases comprising two LAGLIDADG (SEQ ID NO: 55)subunits in which the N-terminal subunit is derived from a naturalmono-LAGLIDADG (SEQ ID NO: 55) meganuclease such as I-CreI, I-MsoI, orI-CeuI or a variant thereof and the C-terminal subunit is derived fromeither a mono-LAGLIDADG (SEQ ID NO: 55) meganuclease or one of the twodomains of a di-LAGLIDADG (SEQ ID NO: 55) meganuclease such as I-SceI,I-DmoI, or I-AniI. The method is distinct from those describedpreviously (Epinat et al. (2003), Nucleic Acids Res. 31: 2952-62;Chevalier et al. (2002), Mol. Cell 10:895-905; WO 2003/078619) in thatit requires the use of specific and novel linker sequences and fusionpoints to produce recombinant single-chain meganucleases in which theN-terminal subunit is derived from a mono-LAGLIDADG (SEQ ID NO: 55)meganuclease.

As described in detail below, the method of producing a recombinantsingle-chain meganuclease includes the use of defined fusion points inthe two LAGLIDADG (SEQ ID NO: 55) subunits to be joined as well as theuse of defined linker sequences to join them into a single polypeptide.In addition, a set of rules is provided for identifying fusion pointsnot explicitly described herein as well as for producing functionallinker sequences that are not explicitly described herein.

Thus, in one aspect, the invention provides methods for producingrecombinant single-chain LAGLIDADG (SEQ ID NO: 55) meganucleases. Inanother aspect, the invention provides the recombinant single-chainmeganucleases resulting from these methods. In another aspect, theinvention provides methods that use such single-chain meganucleases toproduce recombinant nucleic acids, cells and organisms in which adesired DNA sequence or genetic locus within the genome of cell ororganism is modified by the insertion, deletion, substitution or othermanipulation of DNA sequences. In another aspect, the invention providesmethods for reducing the survival of pathogens or cancer cells usingsingle-chain meganucleases which have pathogen-specific orcancer-specific recognition sequences.

1.2 References and Definitions

The patent and scientific literature referred to herein establishesknowledge that is available to those of skill in the art. The issuedU.S. patents, allowed applications, published U.S. and PCT internationalapplications, and references, including GenBank database sequences, thatare cited herein are hereby incorporated by reference to the same extentas if each was specifically and individually indicated to beincorporated by reference.

As used herein, the term “meganuclease” refers to an endonuclease thatbinds double-stranded DNA at a recognition sequence that is greater than12 base pairs in length. Naturally-occurring meganucleases can bemonomeric (e.g., I-SceI) or dimeric (e.g., I-CreI). The termmeganuclease, as used herein, can be used to refer to monomericmeganucleases, dimeric meganucleases, to the monomers which associate toform a dimeric meganuclease, or to a recombinant single-chainmeganuclease of the invention. The term “homing endonuclease” issynonymous with the term “meganuclease.”

As used herein, the term “LAGLIDADG (SEQ ID NO: 55) meganuclease” referseither to meganucleases including a single LAGLIDADG (SEQ ID NO: 55)motif, which are naturally dimeric, or to meganucleases including twoLAGLIDADG (SEQ ID NO: 55) motifs, which are naturally monomeric. Theterm “mono-LAGLIDADG (SEQ ID NO: 55) meganuclease” is used herein torefer to meganucleases including a single LAGLIDADG (SEQ ID NO: 55)motif, and the term “di-LAGLIDADG (SEQ ID NO: 55) meganuclease” is usedherein to refer to meganucleases including two LAGLIDADG (SEQ ID NO: 55)motifs, when it is necessary to distinguish between the two. Each of thetwo structural domains of a di-LAGLIDADG (SEQ ID NO: 55) meganucleasewhich includes a LAGLIDADG (SEQ ID NO: 55) motif and has enzymaticactivity, and each of the individual monomers of a mono-LAGLIDADG (SEQID NO: 55) meganuclease, can be referred to as a LAGLIDADG (SEQ ID NO:55) subunit, or simply “subunit”.

As used herein, and in reference to a peptide sequence, “end” refers tothe C-terminus and “beginning” refers to the N-terminus. Thus, forexample, “the beginning of the LAGLIDADG (SEQ ID NO: 55) motif” refersto the N-terminal-most amino acid in the peptide sequence comprising theLAGLIDADG (SEQ ID NO: 55) motif whereas “the end of the LAGLIDADG (SEQID NO: 55) motif” refers to the C-terminal-most amino acid in thepeptide sequence comprising the LAGLIDADG (SEQ ID NO: 55) motif.

As used herein, the term “rationally-designed” meansnon-naturally-occurring and/or genetically engineered. Therationally-designed meganucleases of the invention differ from wild-typeor naturally-occurring meganucleases in their amino acid sequence orprimary structure, and may also differ in their secondary, tertiary orquaternary structure. In addition, the rationally-designed meganucleasesof the invention also differ from wild-type or naturally-occurringmeganucleases in recognition sequence-specificity and/or activity.

As used herein, with respect to a protein, the term “recombinant” meanshaving an altered amino acid sequence as a result of the application ofgenetic engineering techniques to nucleic acids which encode theprotein, and cells or organisms which express the protein. With respectto a nucleic acid, the term “recombinant” means having an alterednucleic acid sequence as a result of the application of geneticengineering techniques. Genetic engineering techniques include, but arenot limited to, PCR and DNA cloning technologies; transfection,transformation and other gene transfer technologies; homologousrecombination; site-directed mutagenesis; and gene fusion. In accordancewith this definition, a protein having an amino acid sequence identicalto a naturally-occurring protein, but produced by cloning and expressionin a heterologous host, is not considered recombinant.

As used herein with respect to recombinant proteins, the term“modification” means any insertion, deletion or substitution of an aminoacid residue in the recombinant sequence relative to a referencesequence (e.g., a wild-type).

As used herein, the term “genetically-modified” refers to a cell ororganism in which, or in an ancestor of which, a genomic DNA sequencehas been deliberately modified by recombinant technology. As usedherein, the term “genetically-modified” encompasses the term“transgenic.”

As used herein, the term “wild-type” refers to any naturally-occurringform of a meganuclease. The term “wild-type” is not intended to mean themost common allelic variant of the enzyme in nature but, rather, anyallelic variant found in nature. Wild-type meganucleases aredistinguished from recombinant or non-naturally-occurring meganucleases.

As used herein, the term “recognition sequence half-site” or simply“half site” means a nucleic acid sequence in a double-stranded DNAmolecule which is recognized by a monomer of a mono-LAGLIDADG (SEQ IDNO: 55) meganuclease or by one LAGLIDADG (SEQ ID NO: 55) subunit of adi-LAGLIDADG (SEQ ID NO: 55) meganuclease.

As used herein, the term “recognition sequence” refers to a pair ofhalf-sites which is bound and cleaved by either a mono-LAGLIDADG (SEQ IDNO: 55) meganuclease dimer or a di-LAGLIDADG (SEQ ID NO: 55)meganuclease monomer. The two half-sites may or may not be separated bybase pairs that are not specifically recognized by the enzyme. In thecases of I-CreI, I-MsoI and I-CeuI, the recognition sequence half-siteof each monomer spans 9 base pairs, and the two half-sites are separatedby four base pairs which are not contacted directly by binding of theenzyme but which constitute the actual cleavage site (which has a 4 basepair overhang). Thus, the combined recognition sequences of the I-CreI,I-MsoI and I-CeuI meganuclease dimers normally span 22 base pairs,including two 9 base pair half-sites flanking a 4 base pair cleavagesite. In the case of the I-SceI meganuclease, which is a di-LAGLIDADG(SEQ ID NO: 55) meganuclease monomer, the recognition sequence is anapproximately 18 bp non-palindromic sequence, and there are no centralbase pairs which are not specifically recognized. By convention, one ofthe two strands is referred to as the “sense” strand and the other the“antisense” strand, although neither strand may encode protein.

As used herein, the term “specificity” means the ability of ameganuclease to recognize and cleave double-stranded DNA molecules onlyat a particular sequence of base pairs referred to as the recognitionsequence, or only at a particular set of recognition sequences. The setof recognition sequences will share certain conserved positions orsequence motifs, but may be degenerate at one or more positions. Ahighly-specific meganuclease is capable of cleaving only one or a veryfew recognition sequences. Specificity can be determined in a cleavageassay as described in Example 1. As used herein, a meganuclease has“altered” specificity if it binds to and cleaves a recognition sequencewhich is not bound to and cleaved by a reference meganuclease (e.g., awild-type) under physiological conditions, or if the rate of cleavage ofa recognition sequence is increased or decreased by a biologicallysignificant amount (e.g., at least 2×, or 2×-10×) relative to areference meganuclease.

As used herein, the term “palindromic” refers to a recognition sequenceconsisting of inverted repeats of identical half-sites. However, thepalindromic sequence need not be palindromic with respect to the centralbase pairs which are not directly contacted by binding of the enzyme(e.g., the four central base pairs of an I-CreI recognition site). Inthe case of naturally-occurring dimeric meganucleases, palindromic DNAsequences are recognized by homodimers in which the two monomers makecontacts with identical half-sites.

As used herein, the term “pseudo-palindromic” refers to a recognitionsequence consisting of inverted repeats of non-identical or imperfectlypalindromic half-sites. In addition to central base pairs that are notdirectly contacted by binding of the enzyme, the pseudo-palindromicsequence can deviate from a palindromic sequence between the tworecognition half-sites at 1-3 base pairs at each of the two half-sites.Pseudo-palindromic DNA sequences are typical of the natural DNA sitesrecognized by wild-type homodimeric meganucleases in which two identicalenzyme monomers make contacts with slightly different half-sites.

As used herein, the term “non-palindromic” refers to a recognitionsequence composed of two unrelated half-sites of a meganuclease. In thiscase, the non-palindromic sequence need not be palindromic with respectto either the central base pairs or 4 or more base pairs at each of thetwo half-sites. Non-palindromic DNA sequences are recognized by eitherdi-LAGLIDADG (SEQ ID NO: 55) meganucleases, highly degeneratemono-LAGLIDADG (SEQ ID NO: 55) meganucleases (e.g., I-CeuI) or byheterodimers of mono-LAGLIDADG (SEQ ID NO: 55) meganuclease monomersthat recognize non-identical half-sites. In the latter case, anon-palindromic recognition sequence may be referred to as a “hybridsequence” because the heterodimer of two different mono-LAGLIDADG (SEQID NO: 55) monomers, whether or not they are fused into a singlepolypeptide, will cleave a recognition sequence comprising one half-siterecognized by each monomer. Thus, the heterodimer recognition sequenceis a hybrid of the two homodimer recognition sequences.

As used herein, the term “linker” refers to an exogenous peptidesequence used to join two LAGLIDADG (SEQ ID NO: 55) subunits into asingle polypeptide. A linker may have a sequence that is found innatural proteins, or may be an artificial sequence that is not found inany natural protein. A linker may be flexible and lacking in secondarystructure or may have a propensity to form a specific three-dimensionalstructure under physiological conditions.

As used herein, the term “fusion point” refers to the junction between aLAGLIDADG (SEQ ID NO: 55) subunit and a linker. Specifically, the“N-terminal fusion point” is the last (C-terminal-most) amino acid ofthe N-terminal LAGLIDADG (SEQ ID NO: 55) subunit prior to the linkersequence and the “C-terminal fusion point” is the first(N-terminal-most) amino acid of the C-terminal LAGLIDADG (SEQ ID NO: 55)subunit following the linker sequence.

As used herein, the term “single-chain meganuclease” refers to apolypeptide comprising a pair of LAGLIDADG (SEQ ID NO: 55) subunitsjoined by a linker. A single-chain meganuclease has the organization:N-terminal subunit-Linker-C-terminal subunit. A single-chainmeganuclease is distinguished from a natural di-LAGLIDADG (SEQ ID NO:55) meganuclease in that the N-terminal subunit must be derived from amono-LAGLIDADG (SEQ ID NO: 55) meganuclease and, therefore, the linkermust be exogenous to the N-terminal subunit.

As used herein, the term “homologous recombination” refers to thenatural, cellular process in which a double-stranded DNA-break isrepaired using a homologous DNA sequence as the repair template (see,e.g., Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologousDNA sequence may be an endogenous chromosomal sequence or an exogenousnucleic acid that was delivered to the cell. Thus, in some embodiments,a rationally-designed meganuclease is used to cleave a recognitionsequence within a target sequence and an exogenous nucleic acid withhomology to or substantial sequence similarity with the target sequenceis delivered into the cell and used as a template for repair byhomologous recombination. The DNA sequence of the exogenous nucleicacid, which may differ significantly from the target sequence, isthereby incorporated into the chromosomal sequence. The process ofhomologous recombination occurs primarily in eukaryotic organisms. Theterm “homology” is used herein as equivalent to “sequence similarity”and is not intended to require identity by descent or phylogeneticrelatedness.

As used herein, the term “non-homologous end-joining” refers to thenatural, cellular process in which a double-stranded DNA-break isrepaired by the direct joining of two non-homologous DNA segments (see,e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair bynon-homologous end-joining is error-prone and frequently results in theuntemplated addition or deletion of DNA sequences at the site of repair.Thus, in certain embodiments, a rationally-designed meganuclease can beused to produce a double-stranded break at a meganuclease recognitionsequence within a target sequence to disrupt a gene (e.g., byintroducing base insertions, base deletions, or frame-shift mutations)by non-homologous end-joining. In other embodiments, an exogenousnucleic acid lacking homology to or substantial sequence similarity withthe target sequence may be captured at the site of ameganuclease-stimulated double-stranded DNA break by non-homologousend-joining (see, e.g., Salomon et al. (1998), EMBO J. 17:6086-6095).The process of non-homologous end-joining occurs in both eukaryotes andprokaryotes such as bacteria.

As used herein, the term “sequence of interest” means any nucleic acidsequence, whether it codes for a protein, RNA, or regulatory element(e.g., an enhancer, silencer, or promoter sequence), that can beinserted into a genome or used to replace a genomic DNA sequence using ameganuclease protein. Sequences of interest can have heterologous DNAsequences that allow for tagging a protein or RNA that is expressed fromthe sequence of interest. For instance, a protein can be tagged withtags including, but not limited to, an epitope (e.g., c-myc, FLAG) orother ligand (e.g., poly-His). Furthermore, a sequence of interest canencode a fusion protein, according to techniques known in the art (see,e.g., Ausubel et al., Current Protocols in Molecular Biology, Wiley1999). In some embodiments, the sequence of interest is flanked by a DNAsequence that is recognized by the recombinant meganuclease forcleavage. Thus, the flanking sequences are cleaved allowing for properinsertion of the sequence of interest into genomic recognition sequencescleaved by the recombinant meganuclease. In some embodiments, the entiresequence of interest is homologous to or has substantial sequencesimilarity with a target sequence in the genome such that homologousrecombination effectively replaces the target sequence with the sequenceof interest. In other embodiments, the sequence of interest is flankedby DNA sequences with homology to or substantial sequence similaritywith the target sequence such that homologous recombination inserts thesequence of interest within the genome at the locus of the targetsequence. In some embodiments, the sequence of interest is substantiallyidentical to the target sequence except for mutations or othermodifications in the meganuclease recognition sequence such that themeganuclease can not cleave the target sequence after it has beenmodified by the sequence of interest.

As used herein with respect to both amino acid sequences and nucleicacid sequences, the terms “percentage similarity” and “sequencesimilarity” refer to a measure of the degree of similarity of twosequences based upon an alignment of the sequences which maximizessimilarity between aligned amino acid residues or nucleotides, and whichis a function of the number of identical or similar residues ornucleotides, the number of total residues or nucleotides, and thepresence and length of gaps in the sequence alignment. A variety ofalgorithms and computer programs are available for determining sequencesimilarity using standard parameters. As used herein, sequencesimilarity is measured using the BLASTp program for amino acid sequencesand the BLASTn program for nucleic acid sequences, both of which areavailable through the National Center for Biotechnology Information(www.ncbi.nlm.nih.gov/), and are described in, for example, Altschul etal. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), NatureGenet. 3:266-272; Madden et al (1996), Meth. Enzymol. 266:131-141;Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402); Zhang et al.(2000), J. Comput. Biol. 7(1-2):203-14. As used herein, percentsimilarity of two amino acid sequences is the score based upon thefollowing parameters for the BLASTp algorithm: word size=3; gap openingpenalty=−11; gap extension penalty=−1; and scoring matrix=BLOSUM62. Asused herein, percent similarity of two nucleic acid sequences is thescore based upon the following parameters for the BLASTn algorithm: wordsize=11; gap opening penalty=−5; gap extension penalty=−2; matchreward=1; and mismatch penalty=−3.

As used herein with respect to modifications of two proteins or aminoacid sequences, the term “corresponding to” is used to indicate that aspecified modification in the first protein is a substitution of thesame amino acid residue as in the modification in the second protein,and that the amino acid position of the modification in the firstproteins corresponds to or aligns with the amino acid position of themodification in the second protein when the two proteins are subjectedto standard sequence alignments (e.g., using the BLASTp program). Thus,the modification of residue “X” to amino acid “A” in the first proteinwill correspond to the modification of residue “Y” to amino acid “A” inthe second protein if residues X and Y correspond to each other in asequence alignment, and despite the fact that X and Y may be differentnumbers.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable can be equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variablecan be equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable which is described as having values between 0 and 2 can takethe values 0, 1 or 2 if the variable is inherently discrete, and cantake the values 0.0, 0.1, 0.01, 0.001, or any other real values ≥0 and≤2 if the variable is inherently continuous.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

2. Single-Chain Meganucleases Derived from LAGLIDADG (SEQ ID NO: 55)Subunits

Structural comparisons of natural mono- and di-LAGLIDADG (SEQ ID NO: 55)meganucleases reveal that the N-terminal subunits of di-LAGLIDADG (SEQID NO: 55) meganucleases tend to be smaller than mono-LAGLIDADG (SEQ IDNO: 55) monomers. The consequence of this is that, in the case ofdi-LAGLIDADG (SEQ ID NO: 55) meganucleases, the end (C-terminus) of theN-terminal subunit is much closer to the start (N-terminus) of theC-terminal subunit. This means that a relatively short (e.g., 5-20 aminoacid) linker is sufficient to join the two subunits. In the case ofmono-LAGLIDADG (SEQ ID NO: 55) meganucleases, the C-terminus of onemonomer is generally very far (approximately 48 Å in the case of I-CreI)from the N-terminus of the second monomer. Therefore, fusing a pair ofmono-LAGLIDADG (SEQ ID NO: 55) meganucleases into a single polypeptiderequires a longer (e.g., >20 amino acid) peptide linker which can spanthis distance. An alternative method, in which the N-terminal subunit istruncated at a point spatially closer to the start of the C-terminalsubunit has been reported previously (Epinat et al. (2003), NucleicAcids Res. 31: 2952-62; WO 2003/078619), but produces little if anyfunctional heterodimer, as shown in Example 1 below. An extensivediscussion regarding the difficulty associated with producing afunctional single-chain meganuclease derived from I-CreI can be found inFajardo-Sanchez et al. (2008), Nucleic Acids Res. 36:2163-2173.

2.1 Fusion Points for I-CreI

A series of truncation mutants were made in which either wild-typeI-CreI or an engineered variant of I-CreI which had been altered withrespect to its DNA cleavage site preference (designated “CCR2”, SEQ IDNO: 31; see WO 2007/047859) were terminated prior to the naturalC-terminal amino acid, Pro 163 (Table 1). The mutant homodimers wereexpressed in E. coli, purified, and incubated with either the wild-typerecognition sequence (SEQ ID NOs: 34-35) or the CCR2 recognitionsequence (SEQ ID NOs: 32-33) to test for cleavage activity.

TABLE 1 I-CreI Truncation Mutants C-terminal amino acid Wild-typeactivity CCR2 activity Asp-153 + + Val-151 + + Val-148 + − Arg-141 − −Asn-136 − − Val-129 − − Ile-109 − − Leu-95 − −

Wild-type I-CreI was found to be active when truncated at residue 148 orfurther C-terminal residues, but inactive when truncated at residue 141or further N-terminal residues. Therefore, at least some of residues 141through 147, or conservative substitutions of those residues, arerequired for wild-type activity. Similarly, CCR2 was found to be activewhen truncated at residue 151 or further C-terminal residues, butinactive when terminated at residue 148 or further N-terminal residues.Therefore, at least some of residues 148 through 150, or conservativesubstitutions of those residues, are required for CCR2 activity. Thedifference between the wild-type I-CreI and the rationally-designed CCR2meganuclease is presumably due to a reduction in the structuralstability of the CCR2 meganuclease such that it is more sensitive tofurther destabilization by a premature C-terminal truncation. Thesetruncation results are consistent with a publication from Prieto et al.in which it was found that the C-terminal loop of I-CreI (amino acids138-142) is essential for cleavage activity (Prieto et al. (2007), Nucl.Acids Res. 35:3262-3271). Taken together, these results indicate thatsome residues near the C-terminus of I-CreI are essential forDNA-binding and/or catalytic activity and methods for single-chainmeganuclease production that truncate an I-CreI subunit prior toapproximately residue 142 (e.g., Epinat et al. (2003), Nucl. Acids Res.31: 2952-62; WO 2003/078619) are unlikely to yield a single-chainmeganuclease in which both LAGLIDADG (SEQ ID NO: 55) subunits arecatalytically active.

Therefore, in accordance with the present invention, the N-terminalfusion point (i.e., between the N-terminal I-CreI subunit and thelinker) should lie at or C-terminal to residue 142 of the N-terminalsubunit, including any of positions 142-151, or any position C-terminalto residue 151. Residues 154-163 of I-CreI are unstructured (Jurica etal. (1998), Mol. Cell 2:469-476) and, therefore, inclusion of theseresidues will increase the flexibility and, possibly, structuralinstability of the resultant single-chain meganuclease. Conversely, ifit is determined that less flexibility and greater structural stabilityare desired or required, fusion points at residues 142-153 can bechosen.

When the C-terminal LAGLIDADG (SEQ ID NO: 55) subunit of a single-chainmeganuclease is derived from I-CreI, the C-terminal fusion point of thelinker will be toward the N-terminus of the I-CreI sequence. Residues 7,8 and 9 are of particular interest as C-terminal fusion points in I-CreIbecause these residues (1) are structurally conserved among LAGLIDADG(SEQ ID NO: 55) meganuclease family members and, therefore, may providegreater compatibility in forming heterodimers with other LAGLIDADG (SEQID NO: 55) family members, and (2) initiate an alpha-helix containingthe conserved LAGLIDADG (SEQ ID NO: 55) motif that is involved incatalytic function. However, fusion points N-terminal to residue 7,including any of residues 1-6, can also be employed in accordance withthe invention.

The following I-CreI N-terminal and C-terminal fusion points were chosenfor further experimentation, but should not be regarded as limiting thescope of the invention:

TABLE 2 I-CreI Fusion Points N-terminal fusion point C-terminal fusionpoint Val-151 Lys-7 Leu-152 Asp-8 Asp-153 Phe-92.2 Linkers for Single-Chain Meganucleases Derived from I-CreI

For the purpose of linking a pair of I-CreI monomers into a singlepolypeptide, two general classes of linker were considered: (1) anunstructured linker lacking secondary structure; and (2) a structuredlinker having secondary structure. Examples of unstructured linkers arewell known in the art, and include artificial sequences with high Glyand Ser content, or repeats. Structured linkers are also well known inthe art, and include those designed using basic principles of proteinfolding (e.g., Aurora and Rose (1998), Protein Sci. 7:21-38; Fersht,Structure and Mechanism in Protein Science, W.H. Freeman 1998).

The invention was validated using a pair of rationally-designed I-CreImonomers called “LAM1” (SEQ ID NO: 36) and “LAM2” (SEQ ID NO: 37). Theserationally-designed endonucleases were produced using the methodsdescribed in WO 2007/047859 and they are characterized therein. As willbe apparent to those of skill in the art, however, the LAM1 and LAM2monomers are merely exemplary of the many monomers which can beemployed, including wild-type mono-LAGLIDADG (SEQ ID NO: 55) subunits,N-terminally and/or C-terminally truncated wild-type mono-LAGLIDADG (SEQID NO: 55) subunits, N-terminally and/or C-terminally truncatedwild-type di-LAGLIDADG (SEQ ID NO: 55) subunits, and rationally designedmodifications of any of the foregoing.

One exemplary monomer, LAM1, differs by 7 amino acids from wild-typeI-CreI and recognizes the half site:

(SEQ ID NO: 38) 5′-TGCGGTGTC-3′ (SEQ ID NO: 39) 3′-ACGCCACAG-5′Thus, the LAM1 homodimer recognizes the palindromic recognition sequence(where each N is unconstrained):

(SEQ ID NO: 40) 5′-TGCGGTGTCNNNNGACACCGCA-3′ (SEQ ID NO: 41)3′-ACGCCACAGNNNNCTGTGGCGT-5′

The other exemplary monomer. LAM2, differs by 5 amino acids fromwild-type I-CreI and recognizes the half-site:

(SEQ ID NO: 42) 5′-CAGGCTGTC-3′ (SEQ ID NO: 43) 3′-CTCCGACAG-5′Thus, the LAM2 homodimer recognizes the palindromic recognition sequence(where each N is unconstrained):

(SEQ ID NO: 44) 5′-CAGGCTGTCNNNNGACAGCCTG-3′ (SEQ ID NO: 45)3′-GTCCGACAGNNNNCTGTCGGAC-5′

A heterodimer comprising one LAM1 monomer and one LAM2 monomer(“LAM1/LAM2 heterodimer”) thus recognizes the hybrid recognitionsequence:

(SEQ ID NO: 56) 5′-TGCGGTGTCNNNNGACAGCCTG-3′ (SEQ ID NO: 57)3′-ACGCCACAGNNNNCTGTCGGAC-5′

2.2.1 Flexible Linkers for Single-Chain Meganucleases

A variety of highly-flexible peptide linkers are known in the art andcan be used in accordance with the invention. For example, and withoutlimitation, peptide linkers comprising repeating Gly-Ser-Ser units areknown to be unstructured and flexible (Fersht, Structure and Mechanismin Protein Science, W.H. Freeman 1998). Linkers with this and similarcompositions are frequently used to fuse protein domains together (e.g.,single-chain antibodies (Mack et al. (1995), Proc. Nat. Acad. Sci.92:7021-7025); growth factor receptors (Ueda et al. (2000). J. Immunol.Methods 241:159-170); enzymes (Brodelius et al. (2002), 269:3570-3577);and DNA-binding and nuclease domains (Kim et al. (1996), Proc. Nat.Acad. Sci. 93:1156-1160).

As a general matter, the flexible linker can include any polypeptidesequence which does not form stable secondary structures underphysiological conditions. In some embodiments, the linkers include ahigh percentage (e.g., >50%, 60%, 70%, 80% or 90%, or generally,50%-100%) of polar uncharged residues (i.e., Gly, Ser, Cys, Asn, Gin,Tyr, Thr). In addition, in some embodiments, the linkers include a lowpercentage of large hydrophobic residues (i.e., Phe. Trp, Met). Thelinkers may include repeats of varying lengths (e.g., (SG)_(n),(GSS)_(n), (SGGS)_(n) (SEQ ID NO: 58)), may include random sequences, ormay include combinations of the two.

Thus, in accordance with the invention, a set of single-chain fusionsbetween LAM1 and LAM2 were produced in which a highly-flexible peptidelinker covalently joined the N-terminal (LAM1) subunit to the C-terminal(LAM2) subunit using Val-151 or Asp-153 as the N-terminal fusion pointand Phe-9 as the C-terminal fusion point. The single-chain proteins wereexpressed in E. coli, purified, and tested for the ability to cleave ahybrid DNA site comprising one LAM1 half-site and one LAM2 half-site(SEQ ID NOs: 46 and 47). Cleavage activity was rated on a four pointscale: − no detectable activity; + minimal activity; ++ medium activity;+++ activity comparable to the LAM1/LAM2 heterodimer produced byco-expression of the two monomers in E. coli prior to endonucleasepurification. The proteins were also evaluated by SDS-PAGE to determinethe extent to which the linker region was proteolyzed during expressionor purification to release the two subunits.

TABLE 3 Single-Chain I-CreI Meganucleases with Gly-Ser Linkers LinkerN-term. C-term. Linker SEQ ID Linker number fusion pt. fusion pt.sequence NO: Activity proteolysis 1 Val-151 Phe-9 (GSS)₇G 59 - - 2Val-151 Phe-9 (GSS)₈G 60 - - 3 Val-151 Phe-9 (GSS)₉G 61 + + 4 Val-151Phe-9 (GSS)₁₀G 62 ND +++ 5 Val-151 Phe-9 (GSS)₁₁G 63 ND +++ 6 Val-151Phe-9 (GSS)₉GG 64 + + 7 Val-151 Phe-9 (GSS)₉GSG 65 + ++ 8 Asp-153 Phe-9(GSS)₉G 61 + +

The results indicated that flexible linkers, such as the Gly-Ser linkersin Table 3, are suitable for single-chain meganuclease productionprovided that the length is appropriate (see also Example 2). Forexample, referring to Table 3, single-chain meganucleases includinglinkers 1 and 2, comprising 22 and 25 total amino acids, respectively,did not exhibit any detectable cleavage activity with the fusion pointstested. SDS-PAGE indicated that these meganucleases were intact and werenot degraded by proteases, leading to the conclusion that thesesingle-chain meganucleases were structurally stable but functionallyconstrained by linkers that were too short to allow the individualLAGLIDADG (SEQ ID NO: 55) subunits to adopt the necessary conformationfor DNA binding and/or catalysis. Linkers 3, 6, 7, and 8, comprising 28,29, 30, and 28 amino acids, respectively, all exhibited low levels ofcleavage activity. SDS-PAGE indicated that a small amount (5%-10%) ofeach was proteolyzed into individual subunits while the majority had amolecular weight corresponding to the full-length single-chainmeganuclease (˜40 kilodaltons). Numbers 3 and 8 have the same linkersequence but N-terminal fusion points at Val-151 and Asp-153,respectively. Both single-chain meganucleases exhibited comparablelevels of activity, indicating that the precise fusion point is notcritical in this instance. Finally, linkers 4 and 5, comprising 31 and34 amino acids, respectively, yielded no detectable single-chainmeganuclease when purified from E. coli. These linkers were completelyproteolyzed to the individual LAM1 and/or LAM2 subunits as detected bySDS-PAGE and, therefore, the cleavage activity of these meganucleaseswas not investigated further.

These results led us to conclude that Gly-Ser linkers are acceptable forthe production of single-chain meganucleases based upon the LAGLIDADG(SEQ ID NO: 55) subunit of the mono-LAGLIDADG (SEQ ID NO: 55)meganuclease I-CreI and the particular fusion points employed, providedthat the linkers are greater than 25 and less than 31 amino acids inlength. For I-CreI-based single-chain meganucleases with these fusionpoints, shorter linkers prevent catalysis while longer linkers areunstable and prone to clipping by proteases.

The effects of varying the fusions points on the acceptable linkerlengths can be determined empirically by routine experimentation and/orpredicted based upon three-dimensional modeling of the proteinstructures. Significantly, as a fusion point is moved eitherN-terminally or C-terminally, it may move either closer or farther fromthe other fusion point depending upon the secondary and tertiarystructure of the protein near the fusion point. Thus, for example,moving the N-terminal fusion point in the C-terminal direction (e.g.,from residue 150 to residue 155 for an N-terminal subunit) does notnecessarily result in the N-terminal fusion point being physicallycloser to the C-terminal fusion point because, for example, theN-terminal residues in that region may be part of a secondary/tertiarystructure that is pointing either towards or away from the C-terminalfusion point. Thus, moving an N-terminal fusion point in either theN-terminal or C-terminal direction, or moving a C-terminal fusion pointin either the N-terminal or C-terminal direction, can result in a shiftin the range of acceptable linker lengths toward either longer orshorter linkers. That shift, however, is readily determined, as shown bythe experiments reported herein, by routine experimentation and/orthree-dimensional modeling.

Thus, in some embodiments, useful flexible linkers have lengths ofgreater than 25 residues and less than 31 residues (including all valuesin between), as shown in Table 3 for a single-chain meganuclease basedon two I-CreI LAGLIDADG (SEQ ID NO: 55) subunits. In other embodiments,however, employing different LAGLIDADG (SEQ ID NO: 55) subunits and/ordifferent fusion points, useful flexible linkers can have lengthsgreater than 15 and less than 40 residues (including all values inbetween), provided that the linkers are not extensively proteolyzed andthat the single-chain meganuclease retains DNA-binding and cleavageactivity as determined by the simple assays described herein.

2.2.2 Designed, Structured Linkers for Single-Chain Meganucleases

In an effort to produce single-chain I-CreI-based meganucleases withnuclease activity comparable to the natural dimeric enzyme which areboth stable enough for long-term storage and resistant to proteolysis,linkers having stable secondary structures can be used to covalentlyjoin subunits. A search of the Protein Databank (www.rcsb.org) did notreveal any structurally-characterized LAGLIDADG (SEQ ID NO: 55) proteinswith linkers suitable for spanning the great distance (approx. 48 Å)between the identified N- and C-terminal fusion points in I-CreI.Therefore, known first principles governing protein structure (e.g.,Aurora and Rose (1998). Protein Sci. 7:21-38; Fersht, Structure andMechanism in Protein Science, W.H. Freeman 1998) were employed toproduce a set of linkers expected to have structural elements suitablefor joining the two subunits. Specifically, it was postulated that asuitable linker would comprise (listed from N-terminal fusion point toC-terminal fusion point):

(1) Loop 1.

This structural element starts at the N-terminal fusion point andreverses the direction of the peptide chain back on itself (a 180′turn). The sequence can be 3-8 amino acids and can include at least oneglycine residue or, in some embodiments, 2-3 glycines. This structuralelement can be stabilized by introducing a “C-capping” motif toterminate the C-terminal α-helix of I-CreI and initiate the subsequentturn. The helix cap motif is typically described as beginning with ahydrophobic amino acid in the final turn of the helix (Aurora and Rose(1998), Protein Sci. 7:21-38). The C-cap can take any of the formslisted in Table 4:

TABLE 4 C-capping Motifs Number Motif 1 h₁xpx-Gh 2 h₁xpx-nxhx 3h₁xpx-nxph 4 h₁xxx-Gphx 5 h₁xxx-Gpph 6 h₁xxx-Pppph 7 h₁xxx-Ppphwhere h=a hydrophobic amino acid (Ala, Val, Leu, Ile, Met, Phe, Trp,Thr, or Cys); p=a polar amino acid (Gly, Ser, Thr, Asn, Gin, Asp, Glu,Lys, Arg); n=a non-β-branched amino acid (not Val, lie, Thr, or Pro);x=any amino acid from the h or p group; G=glycine; and P=proline. Notethat Thr appears in both groups h and p because its side chain has bothhydrophobic (methyl group) and polar (hydroxyl) functional groups. Thehyphen designates the end of the α-helix and h₁ is a hydrophobic aminoacid in the final turn of the helix (i.e., a hydrophobic amino acid 0-4amino acids prior to the N-terminal fusion point). In the case ofI-CreI, h₁ is typically Val-151 or Leu-152. Thus, an example of motif 7is the sequence V₁₅₁L₁₅₂D₁₅₃S-PGSV (SEQ ID NO: 66) (see, for example,Table 6, Linker 9).

(2) α-Helix 1.

Following Loop 1, this first α-helix in the linker is designed to runanti-parallel to the C-terminal helix in I-CreI (amino acids 144-153) onthe outside face of the protein for a distance of approximately 30 Å.This segment should be 10-20 amino acids in length, should not containany glycine or proline amino acids outside of the N- and C-cappingmotifs (below), and alternate hydrophobic and polar amino acids with 3-4amino acid periodicity so as to bury one face of the helix (thehydrophobic face) against the surface of the N-terminal I-CreI subunitwhile exposing the other face to solvent. The helix could, for example,take the form pphpphhpphpp where p is any polar amino acid and h is anyhydrophobic amino acid but neither is glycine or proline such as thesequence SQASSAASSASS (SEQ ID NO: 67) (see, for example, Table 6, Linker9). Numerous algorithms are available to determine the helicalpropensity of a peptide sequence (e.g., BMERC-PSA,http://bmerc-www.bu.edu/psa/; NNPREDICT,http://alexander.compbio.ucsf.edu/˜nomi/nnpredict.html; PredictProtein,http://wwv.predictprotein.org) and any of these can be used to produce asequence of the appropriate length that can be expected to adoptα-helical secondary structure. Alternatively, this helix sequence couldbe derived from a peptide sequence known to adopt α-helical secondarystructure in an existing natural or designed protein. Numerous examplesof such peptide sequences are available in the Protein Databank(wwwv.rcsb.org).

In addition, it may be desirable to initiate the α-helix with anN-capping motif to stabilize its structure (Aurora and Rose (1998),Protein Sci. 7:21-38). This motif spans the loop-α-helix junction andtypically has one of the forms shown in Table 5:

TABLE 5 N-capping Motifs Number Motif 1 h-xpxhx 2 h-xxpph 3 hp-xpxhx 4hp-xxpph 5 hpp-xpxhx 6 hpp-xxpphwhere the designations are as in Table 4 above and the hyphen representsthe junction between the loop and the helix. An example of motif number2 is the sequence L-SPSQA (SEQ ID NO: 68) (see, for example, Table 6,Linker 9).

(3) Turn 1.

Following α-helix 1, a short, flexible peptide sequence is introduced toturn the overall orientation of the peptide chain by approximately 90relative to the orientation of α-helix 1. This sequence can be 3-8 aminoacids in length and can contain 1 or, in some embodiments, 2-3 glycines.This sequence can also contain a C-cap such as one of the motifs inTable 4 to stabilize α-helix 1 and initiate the turn. An example is thesequence ASSS-PGSGI (SEQ ID NO: 69) (see, for example, Table 6, Linker9) which conforms to C-capping motif number 6. In this case, thesequence ASSS (SEQ ID NO: 70) is the final turn of α-helix 1 while thesequence PGSGI (SEQ ID NO: 71) is Turn 1.

(4) α-Helix 2.

This helix follows Turn 1 and is designed to lie at the surface ofI-CreI in a groove created at the interface between the LAGLIDADG (SEQID NO: 55) subunits. The surface of this groove comprises primarilyamino acids 94-100 and 134-139 of the N-terminal subunit and amino acids48-61 of the C-terminal subunit.

α-helix 2 can be designed to be shorter than α-helix 1 and can comprise1-3 turns of the helix (4-12 amino acids). α-helix 2 can have the sameoverall amino acid composition as α-helix 1 and can also be stabilizedby the addition of an N-capping motif of Table 5. The sequence I-SEALR(SEQ ID NO: 72) is an example (see, for example. Table 6, Linker 9) thatconforms to N-capping motif number 1. Linker 9 incorporates a relativelyshort α-helix 2 comprising the sequence SEALRA (SEQ ID NO: 73) which isexpected to make approximately two turns. Experiments with differentlinker α-helix 2 sequences have demonstrated the importance of helicalregister in this region of the linker. The addition of a single aminoacid (e.g., A, Linker 11), two amino acids (e.g., AS, Linker 12), orthree amino acids (e.g., ASS, Linker 13) prior to the termination ofα-helix 2 with a glycine amino acid can result in single-chain I-CreIproteins that are unstable and precipitate within moments ofpurification from E. coli (Table 6). In contrast, the addition of fouramino acids (e.g., ASSA (SEQ ID NO: 74), linker 14), which is expectedto make one full additional turn and restore the helical register tothat of Linker 1 is stable and active.

(5) Loop 2.

This loop terminates α-helix 2 and turns the peptide chain back onitself to join with the C-terminal I-CreI subunit at the C-terminalfusion point. As with Loop 1, this sequence can be 3-8 amino acids inlength and can contain one or more glycines. It can also contain aC-capping motif from Table 4 to stabilize α-helix 2. For example, thesequence ALRA-GA (SEQ ID NO: 75) from Linker 9 conforms to C-cappingmotif number 1. In addition, this segment can begin an N-cap on theN-terminal α-helix (amino acids 7-20) of the C-terminal I-CreI subunit.For example the sequence T-KSK₇E₈F₉ (SEQ ID NO: 76) from Linker 9conforms to N-capping motif number 2. In this instance, the C-terminalfusion point is Lys-7. In other cases, the fusion point can be movedfurther into the second subunit (for example to amino acids 8 or 9),optionally with the addition of 1-2 amino acids to Loop 2 to compensatefor the change in helical register as the C-terminal fusion point ismoved. For example, linkers 15-23 in Table 6 below have Glu-8 as theC-terminal fusion point and all have an additional amino acid in Loop 2relative to Linkers 1-6.

Employing the principles described above, the set of linkers outlined inTable 6 were developed. A set of single-chain I-CreI meganucleasesincorporating the linkers between LAM1 and LAM2 subunits was constructedand each was tested for activity against the LAM1/LAM2 hybridrecognition sequence. In all cases, the N-terminal fusion point wasAsp-153 of LAM1 and the C-terminal fusion point was either Lys-7 orGlu-8 (denoted in the “CFP” column) of LAM2. Cleavage activity was ratedon a four point scale: − no detectable activity: + minimal activity; ++medium activity; +++ activity comparable to the LAM1/LAM2 heterodimerproduced by co-expression of the two monomers in E. coli prior toendonuclease purification. Immediately following purification, thesingle-chain meganucleases were centrifuged (2100 g for 10 minutes) topellet precipitated protein (indicative of structural instability) andthe amount of precipitate (ppt) observed was scored: − no precipitate: +slight precipitate; ++ significant precipitate. Those protein samplesthat precipitated to a significant degree could not be assayed forcleavage activity.

TABLE 6 Linkers for Single-Chain I-CreI # CFP Linker Sequence SEQ ID NO:Activity ppt  9 K7 SLPGSVGGLSPSQASSAASSASSSPGSGISEA 77 +++ - LRAGATKS 10K7 SLPGSVGGLSPSQASSAASSASSSPGSGISEA 78 +++ - LRAGGATKS 11 K7SLPGSVGGLSPSQASSAASSASSSPGSGISEA 79 ND ++ LRAAGGATKS 12 K7SLPGSVGGLSPSQASSAASSASSSPGSGISEA 80 ND ++ LRAASGGNrKS 13 K7SLPGSVGGLSPSQASSAASSASSSPGSGISEA 81 ND ++ LRAASSGGATKS 14 K7SLPGSVGGLSPSQASSAASSASSSPGSGISEA 82 +++ - LRAASSAGGATKS 15 E8SLPGSVGGLSPSQASSAASSASSSPGSGISEA 83 ++ + LRAGATKEF 16 E8SLPGSVGGISPSQASSAASSASSSPGSGTSEA 84 ++ - PRAGATKEF 17 E8SLPGSVGGLSPSQASSAASSASSSPGSGTSEA 85 ++ + TRAGATKEF 18 E8SLPGSLGGLSPSQASSAASSASSSPGSGPSEA 86 86 + LRAGATKEF 19 E8SLPGSVGGVSPSQASSAASSASSSPGSGVSE 87 ++ + ASRAGATKEF 20 E8SLPGSVGGLSPSQASSAASSASSSPGSGLSEA 88 ++ + LRAGATKEF 21 E8SLPGSLGGISPSQASSAASSASSSPGSGSSEA 89 ++ - SRAGATKEF 22 E8SPGSVGGVSPSQASSAASSASSSPGSGISEAT 90 ++ - RAGATKEF 23 E8SLPGSLGGVSPSQASSAASSPGSGTSEAPRA 91 ND ++ GATKEF 24 E8SLPGSVGGLSPSQASSAASSPGSGISEAIRAG 92 ++ - ATKEF 25 E8SLPGSLGGVSPSQASSAASSASSAASSPGSG 93 ++ - ASEASRAGATKEFSingle-chain meganucleases each of these linkers except for 11-13 and 23(which were not investigated) ran as a single band of the desiredmolecular weight (˜40 kilodaltons) on an SDS-PAGE gel, indicative ofresistance to proteolytic cleavage of the linker sequence. In at leastone case (Linker 9), the single-chain LAM meganuclease could be storedat 4′C in excess of 4 weeks without any evidence of degradation or lossof cleavage activity. Moreover, a number of single-chain LAMendonucleases (9, 10, and 14) cleaved the hybrid LAM1/LAM2 recognitionsequence with efficiency comparable to the purified LAM1/LAM2heterodimer, indicating that fusing I-CreI subunits using these linkersdoes not significantly impair endonuclease activity (see Example 2).

In stark contrast to the purified LAM1/LAM2 heterodimer (which is, infact, a mixture of homo- and heterodimers), the single-chain LAMmeganucleases incorporating the linkers in Table 6 cleave the hybridsite much more efficiently than either of the palindromic sequences (seeExample 2). The palindromic sequences are typically cut with <5%efficiency relative to the hybrid site. This unintended cleavage of thepalindromic DNA sites could be due to (1) homo-dimerization of LAM1 orLAM2 subunits from a pair of different single-chain proteins, (2)sequential nicking of both strands of the palindromic sequence by asingle subunit (LAM1 or LAM2) within the single-chain meganuclease, or(3) minute amounts of homodimeric LAM or LAM2 that form followingproteolytic cleavage of the single-chain meganuclease into itsindividual subunits (although SDS-PAGE results make this latterexplanation unlikely). Although the single-chain I-CreI meganucleasesmaintain some activity toward palindromic DNA sites, the activity is sodisproportionately skewed in favor of the hybrid site that this approachrepresents a very significant improvement over existing methods.

3. Single-Chain Meganucleases Derived from I-MsoI

I-MsoI is a close structural homolog of I-CreI and similar methods havebeen presented for redesigning the DNA-binding specificity of thismeganuclease (WO 2007/047859). The method presented above for theproduction of a single-chain I-CreI meganuclease can be directly appliedto I-MsoI. Amino acids Phe-160, Leu-161, and Lys-162 of I-MsoI arestructurally homologous to, respectively, Val-151, Leu-152, and Asp-153of I-CreI. These amino acids, therefore, can be selected as theN-terminal fusion points for I-MsoI. In addition, The X-ray crystalstructure of I-MsoI reveals that amino acids 161-166 naturally act as aC-cap and initiate a turn at the C-terminus of the protein whichreverses the direction of the peptide chain. Thus, Ile-66 can beselected as the N-terminal fusion point provided that the linker isshortened at its N-terminus to remove the C-cap portion of Loop 1.Pro-9, Thr-10, and Glu-11 of I-MsoI are structurally homologous to,respectively, Lys-7, Glu-8, and Phe-9 of I-CreI and can be selected asC-terminal fusion points for I-MsoI (Table 7). In addition, because thesequence L₇Q₈P₉T₁₀E₁₁A₁₂ (SEQ ID NO: 94) of I-MsoI forms a natural N-cap(motif 2 from Table 5), Leu-7 can be included as a fusion point.

TABLE 7 I-MsoI Fusion Points N-terminal fusion points C-terminal fusionpoints Phe-160 Leu-7 Leu-161 Pro-9 Lys-162 Thr-10 Ile-166 Glu-11

Any of the linkers in Tables 3 or 6 can be used for the production ofsingle-chain I-MsoI endonucleases. For example, Linker 9 from Table 6may be used to join a pair of I-MsoI subunits into a functionalsingle-chain meganuclease using Lys-162 and Pro-9 as fusion points. Inone embodiment, Pro-9 is changed to a different amino acid (e.g.,alanine or glycine) because proline is structurally constraining. Thisis analogous to selecting Thr-10 as the C-terminal fusion point andadding an additional amino acid to the C-terminus of the linkers listedin Tables 3 or 6. For example Linkers 26 and 27 from Table 8 areidentical to Linker 9 from Table 6 except for the addition of a singleamino acid at the C-terminus to account for a change in C-terminalfusion point from Pro-9 (structurally homologous to I-CreI Lys-7) toThr-10 (structurally homologous to I-CreI Glu-8).

In another embodiment, as described in Example 4, a single-chainmeganuclease derived from I-Mso can also be successfully produced usinga linker sequence selected from Linker 28-30 from Table 8 in which I-166is selected as the N-terminal fusion point and Leu-7 is selected as theC-terminal fusion point. Because I-166 is selected as the N-terminalfusion point, the C-cap portion of Loop 1 (corresponding to the first 6amino acids of each of the linkers from Table 6) can be removed. Inaddition, α-helix 1 of Linkers 28-30 are lengthened by 3 amino acids(AAS, underlined in Table 8) relative to the linkers listed in Table 6,corresponding to one additional turn of the helix. Using Linkers 28-30and the specified fusion points, it is possible to produceprotease-resistant, high-activity single-chain meganucleases comprisinga pair of I-Mso-derived subunits (see Example 4).

TABLE 8 Linkers for Single-Chain I-MsoI NFP CFP Linker SequenceSEQ ID NO: Activity ppt 26 K162 T10 PGSVGGLSPSQASSAASSASSSPGSGISEALRA 95++ - GATKSA 27 K162 T10 PGSVGGLSPSQASSAASSASSSPGSGISEALRA 96 ++ - GATKSG28 I166 L7 GGASPSQASSAASSASSAASSPGSGISEALRAA 97 +++ - SSLASKPGST 29 I166L7 GGASPSQASSAASSASSAASSPGSGISEALRAA 98 +++ - SSPGST 30 I166 L7GGASPSQASSAASSASSAASSPGSGPSEALRA 99 +++ - ASSFASKPGST4. Single-Chain Meganucleases Derived from I-CeuI

I-CeuI is a close structural homolog of I-CreI and similar methods havebeen presented for redesigning the DNA-binding specificity of thismeganuclease (WO 2007/047859). The method presented above for theproduction of a single-chain I-CreI meganuclease can be directly appliedto I-CeuI. Amino acids Ala-210, Arg-211, and Asn-212 of I-CeuI arestructurally homologous to, respectively, Val-151, Leu-152, and Asp-153of I-CreI. These amino acids, therefore, can be selected as theN-terminal fusion points for I-CeuI. Ser-53, Glu-54, and Ser-55 ofI-CeuI are structurally homologous to, respectively. Lys-7, Glu-8, andPhe-9 of I-CreI and can be selected as C-terminal fusion points forI-CeuI (Table 9).

TABLE 9 I-CeuI Fusion Points N-terminal fusion points C-terminal fusionpoints Ala-210 Ser-53 Arg-211 Glu-54 Asn-212 Ser-55

Any of the linkers in Tables 3 or 6 can be effective for the productionof single-chain I-CeuI endonucleases. For example, I-CeuI subunits canbe joined by Linker 9 from Table 6 using Asn-212 as the N-terminalfusion point and Ser-53 as the C-terminal fusion point.

The C-terminal fusion points selected for I-CeuI result in the removalof amino acids 1-52 from the C-terminal I-CeuI subunit. Structuralanalyses (Spiegel et al. (2006), Structure 14:869-880) reveal that theseamino acids form a structured domain that rests on the surface of I-CeuIand buries a substantial amount of hydrophobic surface area contributedby amino acids 94-123. It is possible, therefore, that removing thisN-terminal domain will destabilize the C-terminal I-CeuI subunit in thesingle-chain meganuclease. To mitigate this possibility, the hydrophobicamino acids that would be exposed by the removal of this N-terminaldomain can be mutated to polar amino acids (e.g. non-β-branched,hydrophobic amino acids can be mutated to Ser while β-branched,hydrophobic amino acids can be mutated to Thr). For example, Leu-101,Tyr-102, Leu-105, Ala-121, and Leu-123 can be mutated to Ser whileVal-95, Val-98, and lie-113 can be mutated to Thr.

Alternatively, the N-terminal domain of the C-terminal I-CeuI subunitcan be left largely intact and joined to the N-terminal subunit via atruncated linker. This can be accomplished using Lys-7, Pro-8, Gly-9, orGlu-10 (SEQ ID NO: 100) as a C-terminal fusion point. The linker can bea flexible Gly-Ser linker (e.g., Linker 3 from Table 3) truncated byapproximately 50% of its length (i.e., (GSS)₄G (SEQ ID NO: 101) or(GSS)₅G (SEQ ID NO: 102)). Alternatively, the linker can be any of thelinkers from Table 6 truncated within Turn 1. Thus, using Linker 9 fromTable 6 as an example, a single-chain I-CeuI meganuclease can be madewith the following composition:

N-term. subunit N₂₁₂- (SEQ ID NO: 103)SLPGSVGGLSPSQASSAASSASSSPGS-G₉ C-term. subunit5. Single-Chain Meganucleases Derived from Two Different LAGLIDADG (SEQID NO: 55) Family Members

This invention also enables the production of single-chain meganucleasesin which each of the subunits is derived from a different naturalLAGLIDADG (SEQ ID NO: 55) domain. “Different,” as used in thisdescription, refers to LAGLIDADG (SEQ ID NO: 55) subunits that are notderived from the same natural LAGLIDADG (SEQ ID NO: 55) family member.Thus, as used in this description, rationally-designed LAGLIDADG (SEQ IDNO: 55) subunits from the same family member (e.g., two I-CreI subunitsthat have been genetically engineered with respect to DNA cleavagespecificity) are not considered “different”. Specifically, the inventionenables the production of single-chain meganucleases comprising anN-terminal subunit derived from a mono-LAGLIDADG (SEQ ID NO: 55)meganuclease (e.g., I-CreI, I-MsoI, or I-CeuI) linked to a C-terminalsubunit derived from a different mono-LAGLIDADG (SEQ ID NO: 55)meganuclease or either of the two LAGLIDADG (SEQ ID NO: 55) domains froma di-LAGLIDADG (SEQ ID NO: 55) meganuclease. For example, a single-chainmeganuclease can be produced comprising an N-terminal I-CreI subunit,which may or may not have been rationally-designed with regard to DNArecognition site specificity, linked to a C-terminal I-MsoI subunitwhich also may or may not have been rationally-designed with regard toDNA recognition site specificity.

In the cases of I-CreI, I-MsoI, and I-CeuI, the desirable fusion pointsand linkers are as described above. For example, a single-chain I-CreIto I-MsoI fusion can be produced using Linker 9 from Table 6 to joinI-CreI Asp-153 to I-MsoI Thr-10. Table 9 lists potential C-terminalfusion points for individual LAGLIDADG (SEQ ID NO: 55) domains from thedi-LAGLIDADG (SEQ ID NO: 55) meganucleases I-SceI, I-DmoI, and I-AniI.

TABLE 10 C-terminal Fusion Points for di-LAGLIDADG (SEQ ID NO: 55)Meganuclease Subunits I-SceI I-SceI I-AniI I-AniI I-DmoI I-DmoIN-terminal C-terminal N-terminal C-terminal N-terminal C-terminal(31-123) (132-225) (3-125) (135-254) (8-98) (104-178) I-31 Y-132 D3S-135 S-8 R-104 E-32 L-133 L4 Y-136 G-9 E-105 Q-33 T-134 Y6 F-137 I-10Q-106The fusion points listed in Tables 7, 9 and 10 are based on structurecomparisons between the meganuclease in question and I-CreI in whichamino acid positions which are structurally homologous to the I-CreIfusion points were identified. Fusion points can also be identified inLAGLIDADG (SEQ ID NO: 55) subunits which have not been structurallycharacterized using protein sequence alignments to I-CreI. This isparticularly true of C-terminal fusion points which can be readilyidentified in any LAGLIDADG (SEQ ID NO: 55) subunit based upon thelocation of the conserved LAGLIDADG (SEQ ID NO: 55) motif. The aminoacids which are 4-6 residues N-terminal of the start of the LAGLIDADG(SEQ ID NO: 55) motif are acceptable C-terminal fusion points.

Because the dimerization interfaces between subunits from differentLAGLIDADG (SEQ ID NO: 55) endonucleases vary, the subunits may notassociate into functional “heterodimers” despite being covalently joinedas a single polypeptide. To promote association, the interface betweenthe two subunits can be rationally-designed, as described in WO2007/047859. At its simplest, this involves substituting interfaceresidues from one subunit onto another. For example, I-CreI and I-MsoIdiffer in the interface region primarily at I-CreI Glu-8 (which is a Thrin the homologous position of I-MsoI, amino acid 10) and Leu-11 (whichis an Ala in the homologous position of I-MsoI, amino acid 13). Thus,I-CreI and I-MsoI subunits can be made to interact effectively bychanging Glu-8 and Leu-11 of the I-CreI subunit to Thr and Ala,respectively, or by changing Thr-10 and Ala-13 of the I-MsoI subunit toGlu and Leu, respectively.

Techniques such as computational protein design algorithms can also beused to rationally-design the subunit interfaces. Such methods are knownin the art. For example, Chevalier et al. used a computational algorithmto redesign the interface between I-CreI and the N-terminal LAGLIDADG(SEQ ID NO: 55) domain of I-DmoI to enable the two to interact(Chevalier et al. (2002), Mol. (ell 10:895-905). Taking these resultsinto account, a single-chain meganuclease comprising an N-terminalsubunit derived from I-CreI and a C-terminal subunit derived from theN-terminal LAG[[A]]LIDADG (SEQ ID NO: 55) domain of I-DmoI can beproduced by (1) selecting an N-terminal fusion point in I-CreI fromTable 2, (2) selecting a C-terminal fusion point in I-DmoI from Table10, (3) selecting a linker from Table 6 (or designing a similar linkerbased on the rules provided), and (4) incorporating the mutations L11A,F161, K96N, and L97F into the I-CreI subunit and the mutations I19W,H51F, and L55R into the I-DmoI subunit as proposed by Chevalier et al.

Alternatively, empirical methods such as directed evolution can be usedto engineer the interface between two different LAGLIDADG (SEQ ID NO:55) subunits. Such methods are known in the art. For example, geneticlibraries can be produced in which specific amino acids in the subunitinterface are randomized, and library members which permit theinteraction between the two subunits are screened experimentally. Suchscreening methods are known in the art (e.g., Sussman et al. (2004), JMol. Biol. 342: 31-41, Chames et al. (2005), Nucl. Acids Res. 33: e178;Seligman et al. (2002), Nucl. Acids Res. 30: 3870-9, Amould et al.(2006), J. Mol. Biol. 355: 443-58) and can be conducted to test for theability of a single-chain meganuclease comprising two differentLAGLIDADG (SEQ ID NO: 55) subunits to cleave a hybrid DNA site within ayeast or bacterial cell.

6. Single-Chain Meganucleases with Altered DNA-Cleavage Specificity,Activity, and/or DNA-Binding Affinity

The invention can be used to produce single-chain meganucleasescomprising individual LAGLIDADG (SEQ ID NO: 55) subunits that have beengenetically-engineered with respect to DNA-cleavage specificity using avariety of methods. Such methods include rational-design (e.g., WO2007/047859), computational design (e.g., Ashworth et al. (2006), Nature441:656-659), and genetic selection (Sussman et al. (2004), J. Mol.Biol. 342; 31-41; Chames et al. (2005), Nucl. Acids Res. 33: e178:Seligman et al. (2002), Nucl. Acids Res. 30: 3870-9. Amould et al.(2006), J. Mol. Biol. 355: 443-58). Such meganucleases can be targetedto DNA sites that differ from the sites recognized by wild-typemeganucleases. The invention can also be used to join LAGLIDADG (SEQ IDNO: 55) subunits that have been rationally-designed to have alteredactivity (e.g., WO 2007/047859; Amould et al. (2007), J. Mol. Biol371(1):49-65) or DNA-binding affinity as described in WO 2007/047859.

7. Methods of Producing Recombinant Cells and Organisms

Aspects of the present invention further provide methods for producingrecombinant, transgenic or otherwise genetically-modified cells andorganisms using single-chain meganucleases. Thus, in certainembodiments, recombinant single-chain meganucleases are developed tospecifically cause a double-stranded break at a single site or atrelatively few sites in the genomic DNA of a cell or an organism toallow for precise insertion(s) of a sequence of interest by homologousrecombination. In other embodiments, recombinant meganucleases aredeveloped to specifically cause a double-stranded break at a single siteor at relatively few sites in the genomic DNA of a cell or an organismto either (a) allow for rare insertion(s) of a sequence of interest bynon-homologous end-joining or (b) allow for the disruption of the targetsequence by non-homologous end-joining. As used herein with respect tohomologous recombination or non-homologous end-joining of sequences ofinterest, the term “insertion” means the ligation of a sequence ofinterest into a chromosome such that the sequence of interest isintegrated into the chromosome. In the case of homologous recombination,an inserted sequence can replace an endogenous sequence, such that theoriginal DNA is replaced by exogenous DNA of equal length, but with analtered nucleotide sequence. Alternatively, an inserted sequence caninclude more or fewer bases than the sequence it replaces.

Therefore, in accordance with this aspect of the invention, therecombinant organisms include, but are not limited to, monocot plantspecies such as rice, wheat, corn (maize) and rye, and dicot speciessuch as legumes (e.g., kidney beans, soybeans, lentils, peanuts, peas),alfalfa, clover, tobacco and Arabidopsis species. In addition, therecombinant organisms can include, but are not limited to, animals suchas humans and non-human primates, horses, cows, goats, pigs, sheep,dogs, cats, guinea pigs, rats, mice, lizards, fish and insects such asDrosophila species. In other embodiments, the organism is a fungus suchas a Candida, Neurospora or Saccharomyces species.

In some embodiments, the methods of the invention involve theintroduction of a sequence of interest into a cell such as a germ cellor stem cell that can become a mature recombinant organism or allow theresultant genetically-modified organism to give rise to progeny carryingthe inserted sequence of interest in its genome.

Meganuclease proteins can be delivered into cells to cleave genomic DNA,which allows for homologous recombination or non-homologous end-joiningat the cleavage site with a sequence of interest, by a variety ofdifferent mechanisms known in the art. For example, the recombinantmeganuclease protein can introduced into a cell by techniques including,but not limited to, microinjection or liposome transfections (see, e.g.,Lipofectamine™, Invitrogen Corp., Carlsbad, Calif.). The liposomeformulation can be used to facilitate lipid bilayer fusion with a targetcell, thereby allowing the contents of the liposome or proteinsassociated with its surface to be brought into the cell. Alternatively,the enzyme can be fused to an appropriate uptake peptide such as thatfrom the HIV TAT protein to direct cellular uptake (see, e.g., Hudecz etal. (2005), Med. Res. Rev. 25: 679-736).

Alternatively, gene sequences encoding the meganuclease protein areinserted into a vector and transfected into a eukaryotic cell usingtechniques known in the art (see, e.g., Ausubel et. al., CurrentProtocols in Molecular Biology, Wiley 1999). The sequence of interestcan be introduced in the same vector, a different vector, or by othermeans known in the art.

Non-limiting examples of vectors for DNA transfection include virusvectors, plasmids, cosmids, and YAC vectors. Transfection of DNAsequences can be accomplished by a variety of methods known to those ofskill in the art. For instance, liposomes and immunoliposomes are usedto deliver DNA sequences to cells (see, e.g., Lasic et al. (1995),Science 267: 1275-76). In addition, viruses can be utilized to introducevectors into cells (see, e.g., U.S. Pat. No. 7,037,492). Alternatively,transfection strategies can be utilized such that the vectors areintroduced as naked DNA (see, e.g., Rui et al. (2002), Life Sci. 71(15):1771-8).

General methods for delivering nucleic acids into cells include: (1)chemical methods (Graham et al. (1973), Virology 54(2):536-539;Zatloukal et al. (1992), Ann. N. Y. Acad Sci., 660:136-153; (2) physicalmethods such as microinjection (Capecchi (1980), Cell 22(2):479-488,electroporation (Wong et al. (1982). Biochim. Biophys. Res. Commun.107(2):584-587; Fromm et al. (1985), Proc. Nat'l Acad. Sci. USA82(17):5824-5828; U.S. Pat. No. 5,384,253) and ballistic injection(Johnston et al. (1994), Methods Cell. Biol. 43(A): 353-365; Fynan etal. (1993), Proc. Nat'l Acad. Sci. USA 90(24): 11478-11482); (3) viralvectors (Clapp (1993), Clin. Perinatol. 20(1): 155-168; Lu et al.(1993). J. Exp. Med. 178(6):2089-2096: Eglitis et al. (1988), Avd. Exp.Med. Biol. 241:19-27; Eglitis et al. (1988). Biotechniques6(7):608-614); and (4) receptor-mediated mechanisms (Curiel et al.(1991), Proc. Nat'l Acad. Sci. (SA 88(19):8850-8854: Curiel et al.(1992), Hum. Gen. Ther. 3(2):147-154: Wagner et al. (1992), Proc. Nat'lAcad. Sci. USA 89 (13):6099-6103).

In certain embodiments, a genetically-modified plant is produced, whichcontains the sequence of interest inserted into the genome. In certainembodiments, the genetically-modified plant is produced by transfectingthe plant cell with DNA sequences corresponding to the recombinantmeganuclease and the sequence of interest, which may or may not beflanked by the meganuclease recognition sequences and/or sequencessubstantially identical to the target sequence. In other embodiments,the genetically-modified plant is produced by transfecting the plantcell with DNA sequences corresponding to the recombinant meganucleaseonly, such that cleavage promotes non-homologous end-joining anddisrupts the target sequence containing the recognition sequence. Insuch embodiments, the meganuclease sequences are under the control ofregulatory sequences that allow for expression of the meganuclease inthe host plant cells. These regulatory sequences include, but are notlimited to, constitutive plant promoters such as the NOS promoter,chemically-inducible gene promoters such as the dexamethasone-induciblepromoter (see, e.g., Gremillon et al. (2004), Plant J. 37:218-228), andplant tissue specific promoters such as the LGC1 promoter (see, e.g.,Singh et al. (2003), FEBS Lett. 542:47-52).

Suitable methods for introducing DNA into plant cells include virtuallyany method by which DNA can be introduced into a cell, including but notlimited to Agrobacterium infection, PEG-mediated transformation ofprotoplasts (Omirulleh et al. (1993), Plant Molecular Biology,21:415-428), desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers, ballisticinjection or microprojectile bombardment, and the like.

In other embodiments, a genetically-modified animal is produced using arecombinant meganuclease. As with plant cells, the nucleic acidsequences can be introduced into a germ cell or a cell that willeventually become a transgenic organism. In some embodiments, the cellis a fertilized egg, and exogenous DNA molecules can be injected intothe pro-nucleus of the fertilized egg. The micro-injected eggs are thentransferred into the oviducts of pseudopregnant foster mothers andallowed to develop. The recombinant meganuclease is expressed in thefertilized egg (e.g., under the control of a constitutive promoter, suchas 3-phosphoglycerate kinase), and facilitates homologous recombinationof the sequence of interest into one or a few discrete sites in thegenome. Alternatively, the genetically-modified animals can be obtainedby utilizing recombinant embryonic stem (“ES”) cells for the generationof the transgenics, as described by Gossler et al. (1986), Proc. Natl.Acad. Sci. USA 83:9065 9069.

In certain embodiments, a recombinant mammalian expression vector iscapable of directing tissue-specific expression of the nucleic acidpreferentially in a particular cell type. Tissue-specific regulatoryelements are known in the art. Non-limiting examples of suitabletissue-specific promoters include the albumin promoter (liver-specific;Pinkert et al. (1987), Genes Dev. 1: 268-277), lymphoid-specificpromoters (Calame and Eaton (1988), Adv. Immunol. 43: 235-275), inparticular promoters of T cell receptors (Winoto and Baltimore (1989),EMBO. J. 8: 729-733) and immunoglobulins (Banerji et al. (1983), Cell33: 729-740; Queen and Baltimore (1983), Cell 33: 741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989), Proc. Natl. Acad. Sci. USA 86: 5473-5477),pancreas-specific promoters (Edlund et al. (1985), Science 230:912-916), and mammary gland-specific promoters (e.g., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Pat. Pub. EP 0 264 166).Developmentally-regulated promoters are also encompassed, e.g., themurine hox promoters (Kessel and Gruss (1990), Science 249: 374-379) andthe α-fetoprotein promoter (Campes and Tilghman (1989), Genes Dev. 3:537-546).

In certain embodiments, a single-chain meganuclease may be tagged with apeptide epitope (e.g., an HA, FLAG, or Myc epitope) to monitorexpression levels or localization. In some embodiments, the meganucleasemay be fused to a sub-cellular localization signal such as anuclear-localization signal (e.g., the nuclear localization signal fromSV40) or chloroplast or mitochondrial localization signals. In otherembodiments, the meganuclease may be fused to a nuclear export signal tolocalize it to the cytoplasm. The meganuclease may also be fused to anunrelated protein or protein domain such as a protein that stimulatesDNA-repair or homologous recombination (e.g., recA, RAD51, RAD52, RAD54,RAD57 or BRCA2).

8. Methods for Gene Therapy

Aspects of the invention allow for the use of recombinant meganucleasefor gene therapy. As used herein, “gene therapy” means therapeutictreatments that comprise introducing into a patient a functional copy ofat least one gene, or gene regulatory sequence such as a promoter,enhancer, or silencer to replace a gene or gene regulatory region thatis defective in its structure and/or function. The term “gene therapy”can also refer to modifications made to a deleterious gene or regulatoryelement (e.g., oncogenes) that reduce or eliminate expression of thegene. Gene therapy can be performed to treat congenital conditions,conditions resulting from mutations or damage to specific genetic lociover the life of the patient, or conditions resulting from infectiousorganisms.

In some aspects of the invention, dysfunctional genes are replaced ordisabled by the insertion of exogenous nucleic acid sequences into aregion of the genome affecting gene expression. In certain embodiments,the recombinant meganuclease is targeted to a particular sequence in theregion of the genome to be modified so as to alleviate the condition.The sequence can be a region within an exon, intron, promoter, or otherregulatory region that is causing dysfunctional expression of the gene.As used herein, the term “dysfunctional expression” means aberrantexpression of a gene product either by the cell producing too little ofthe gene product, too much of the gene product, or producing a geneproduct that has a different function such as lacking the necessaryfunction or having more than the necessary function.

Exogenous nucleic acid sequences inserted into the modified region canbe used to provide “repaired” sequences that normalize the gene. Generepair can be accomplished by the introduction of proper gene sequencesinto the gene allowing for proper function to be reestablished. In theseembodiments, the nucleic acid sequence to be inserted can be the entirecoding sequence for a protein or, in certain embodiments, a fragment ofthe gene comprising only the region to be repaired. In other embodimentsthe nucleic acid sequence to be inserted comprises a promoter sequenceor other regulatory elements such that mutations causing abnormalexpression or regulation are repaired. In other embodiments, the nucleicacid sequence to be inserted contains the appropriate translation stopcodon lacking in a mutated gene. The nucleic acid sequence can also havesequences for stopping transcription in a recombinant gene lackingappropriate transcriptional stop signals.

Alternatively, the nucleic acid sequences can eliminate gene functionaltogether by disrupting the regulatory sequence of the gene orproviding a silencer to eliminate gene function. In some embodiments,the exogenous nucleic acid sequence provides a translation stop codon toprevent expression of the gene product. In other embodiments, theexogenous nucleic acid sequences provide transcription stop element toprevent expression of a full length RNA molecule. In still otherembodiments, gene function is disrupted directly by the meganuclease byintroducing base insertions, base deletions, and/or frameshift mutationsthrough non-homologous end-joining.

In many instances, it is desirable to direct the proper geneticsequences to a target cell or population of cells that is the cause ofthe disease condition. Such targeting of therapeutics prevents healthycells from being targeted by the therapeutics. This increases theefficacy of the treatment, while decreasing the potentially adverseeffects that the treatment could have on healthy cells.

Delivery of recombinant meganuclease genes and the sequence of interestto be inserted into the genome to the cells of interest can beaccomplished by a variety of mechanisms. In some embodiments, thenucleic acids are delivered to the cells by way of viruses withparticular viral genes inactivated to prevent reproduction of the virus.Thus, a virus can be altered so that it is capable only of delivery andmaintenance within a target cell, but does not retain the ability toreplicate within the target cell or tissue. One or more DNA sequencescan be introduced to the altered viral genome, so as to produce a viralgenome that acts like a vector, and may or may not be inserted into ahost genome and subsequently expressed. More specifically, certainembodiments include employing a retroviral vector such as, but notlimited to, the MFG or pLJ vectors. An MFG vector is a simplifiedMoloney murine leukemia virus vector (MoMLV) in which the DNA sequencesencoding the pol and env proteins have been deleted to render itreplication defective. A pLJ retroviral vector is also a form of theMoMLV (see, e.g., Korman et al. (1987), Proc. Nat'l Acad. Sci.,84:2150-2154). In other embodiments, a recombinant adenovirus oradeno-associated virus can be used as a delivery vector.

In other embodiments, the delivery of recombinant meganuclease proteinand/or recombinant meganuclease gene sequences to a target cell isaccomplished by the use of liposomes. The production of liposomescontaining nucleic acid and/or protein cargo is known in the art (see,e.g., Lasic et al. (1995), Science 267: 1275-76). Immunoliposomesincorporate antibodies against cell-associated antigens into liposomes,and can delivery DNA sequences for the meganuclease or the meganucleaseitself to specific cell types (see, e.g., Lasic et al. (1995), Science267: 1275-76; Young et al. (2005), J. Calif. Dent. Assoc. 33(12):967-71; Pfeiffer et al. (2006), J. Vasc. Surg. 43(5): 1021-7). Methodsfor producing and using liposome formulations are well known in the art,(see, e.g., U.S. Pat. No. 6,316,024, U.S. Pat. No. 6,379,699, U.S. Pat.No. 6,387,397, U.S. Pat. No. 6,511,676 and U.S. Pat. No. 6,593,308, andreferences cited therein). In some embodiments, liposomes are used todeliver the sequences of interest as well as the recombinantmeganuclease protein or recombinant meganuclease gene sequences.

9. Methods for Treating Pathogen Infection.

Aspects of the invention also provide methods of treating infection by apathogen. Pathogenic organisms include viruses such as, but not limitedto, herpes simplex virus 1, herpes simplex virus 2, humanimmunodeficiency virus 1, human immunodeficiency virus 2, variola virus,polio virus, Epstein-Barr virus, and human papilloma virus and bacterialorganisms such as, but not limited to, Bacillus anthracis, Haemophilusspecies, Pneumococcus species, Staphylococcus aureus, Streptococcusspecies, methicillin-resistant Staphylococcus aureus, and Mycoplasmatuberculosis. Pathogenic organisms also include fungal organisms suchas, but not limited to, Candida, Blastomyces, Cryptococcus, andHistoplasma species.

In some embodiments, a single-chain meganuclease can be targeted to arecognition sequence within the pathogen genome, e.g., to a gene orregulatory element that is essential for growth, reproduction, ortoxicity of the pathogen. In certain embodiments, the recognitionsequence may be in a bacterial plasmid. Meganuclease-mediated cleavageof a recognition sequence in a pathogen genome can stimulate mutationwithin a targeted, essential gene in the form of an insertion, deletionor frameshift, by stimulating non-homologous end-joining. Alternatively,cleavage of a bacterial plasmid can result in loss of the plasmid alongwith any genes encoded on it, such as toxin genes (e.g., B. anthracisLethal Factor gene) or antibiotic resistance genes. As noted above, themeganuclease may be delivered to the infected patient, animal, or plantin either protein or nucleic acid form using techniques that are commonin the art. In certain embodiments, the meganuclease gene may beincorporated into a bacteriophage genome for delivery to pathogenicbacteria.

Aspects of the invention also provide therapeutics for the treatment ofcertain forms of cancer. Because human viruses are often associated withtumor formation (e.g., Epstein-Barr Virus and nasopharyngeal carcinomas;Human Papilloma Virus and cervical cancer) inactivation of these viralpathogens may prevent cancer development or progression. Alternatively,double-stranded breaks targeted to the genomes of these tumor-associatedviruses using single-chain meganucleases may be used to triggerapoptosis through the DNA damage response pathway. In this manner, itmay be possible to selectively induce apoptosis in tumor cells harboringthe viral genome.

10. Methods for Genotyping and Pathogen Identification

Aspects of the invention also provide tools for in vitro molecularbiology research and development. It is common in the art to usesite-specific endonucleases (e.g., restriction enzymes) for theisolation, cloning, and manipulation of nucleic acids such as plasmids,PCR products, BAC sequences, YAC sequences, viruses, and genomicsequences from eukaryotic and prokaryotic organisms (see, e.g., Ausubelet al., Current Protocols in Molecular Biology. Wiley 1999). Thus, insome embodiments, a single-chain meganuclease may be used to manipulatenucleic acid sequences in vitro. For example, single-chain meganucleasesrecognizing a pair of recognition sequences within the same DNA moleculecan be used to isolate the intervening DNA segment for subsequentmanipulation such as ligation into a bacterial plasmid, BAC, or YAC.

In another aspect, this invention provides tools for the identificationof pathogenic genes and organisms. In one embodiment, single-chainmeganucleases can be used to cleave recognition sites corresponding topolymorphic genetic regions correlated to disease to distinguishdisease-causing alleles from healthy alleles (e.g., a single-chainmeganuclease which recognizes the ΔF-508 allele of the human CFTR gene,see example 4). In this embodiment, DNA sequences isolated from a humanpatient or other organism are digested with a single-chain meganuclease,possibly in conjunction with additional site-specific nucleases, and theresulting DNA fragment pattern is analyzed by gel electrophoresis,capillary electrophoresis, mass spectrometry, or other methods known inthe art. This fragmentation pattern and, specifically, the presence orabsence of cleavage by the single-chain meganuclease, indicates thegenotype of the organism by revealing whether or not the recognitionsequence is present in the genome. In another embodiment, a single-chainmeganuclease is targeted to a polymorphic region in the genome of apathogenic virus, fungus, or bacterium and used to identify theorganism. In this embodiment, the single-chain meganuclease cleaves arecognition sequence that is unique to the pathogen (e.g., the spacerregion between the 16S and 23S rRNA genes in a bacterium; see, e.g., vander Giessen et al. (1994). Microbiology 140:1103-1108) and can be usedto distinguish the pathogen from other closely-related organismsfollowing endonuclease digest of the genome and subsequent analysis ofthe fragmentation pattern by electrophoresis, mass spectrometry, orother methods known in the art.

11. Methods for the Production of Custom DNA-Binding Domains.

In another aspect, the invention provides single-chain DNA-bindingproteins that lack endonuclease cleavage activity. The catalyticactivity of a single-chain meganuclease can be eliminated by mutatingamino acids involved in catalysis (e.g., the mutation of Q47 to E inI-CreI, see Chevalier et al. (2001), Biochemistry. 43:14015-14026); themutation of D44 or D145 to N in I-SceI; the mutation of E66 to Q inI-CeuI; the mutation of D22 to N in I-MsoI). The inactivatedmeganuclease can then be fused to an effector domain from anotherprotein including, but not limited to, a transcription activator (e.g.,the GAL4 transactivation domain or the VP16 transactivation domain), atranscription repressor (e.g., the KRAB domain from the Kruppelprotein), a DNA methylase domain (e.g., M.CviPI or M.SssI), or a histoneacetyltransferase domain (e.g., HDAC1 or HDAC2). Chimeric proteinsconsisting of an engineered DNA-binding domain, most notably anengineered zinc finger domain, and an effector domain are known in theart (see, e.g., Papworth et al. (2006). Gene 366:27-38).

EXAMPLES

This invention is further illustrated by the following examples, whichshould not be construed as limiting. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific substances andprocedures described herein. Such equivalents are intended to beencompassed in the scope of the claims that follow the examples below.Example 1 presents evidence that a previously disclosed method for theproduction of single-chain I-CreI meganucleases (Epinat et al. (2003),Nucleic Acids Res. 31: 2952-62; WO 2003/078619) is not sufficient forthe production of meganucleases recognizing non-palindromic DNA sites.Examples 2 and 3 present evidence that the method described here issufficient to produce single-chain I-CreI meganucleases recognizingnon-palindromic DNA sites using a flexible Gly-Ser linker (example 2) ora designed, structured linker (example 3). Although examples 2 and 3below refer specifically to single-chain meganucleases based on I-CreI,single-chain meganucleases comprised of subunits derived from I-SceI,I-MsoI, I-CeuI, I-AniI, and other LAGLIDADG (SEQ ID NO: 55)meganucleases can be similarly produced and used, as described herein.

Example 1 Evaluation of the Method of Epinat et al 1. Single ChainMeganucleases Using the Method of Epinat et al.

Epinat et al. (2003), Nucleic Acids Res. 31: 2952-62 and WO 2003/078619report the production of a single-chain meganuclease derived from theI-CreI meganuclease. Specifically, the authors used an 11 amino acidpeptide linker derived from I-DmoI (amino acids 94-104 of I-DmoI,sequence MLERIRLFNMR (SEQ ID NO: 104)) to join an N-terminal I-CreIsubunit (amino acids 1-93 of I-CreI) to a C-terminal I-CreI subunit(amino acids 8-163). This particular arrangement of N-terminalsubunit-linker-C-terminal subunit was selected because it most closelymimics the domain organization of the di-LAGLIDADG (SEQ ID NO: 55)I-DmoI meganuclease. The authors evaluated the single-chain I-CreImeganuclease experimentally and found it to cleave a wild-type I-CreIrecognition sequence effectively, albeit at a significantly reduced raterelative to the wild-type I-CreI homodimer.

Because the fusion protein produced by these authors comprised twootherwise wild-type subunits, both of which recognize identical DNAhalf-sites, it was necessary to test the single-chain meganuclease usingthe pseudo-palindromic wild-type DNA site. As such, it was not possiblefor the authors to rule out the possibility that the observed cleavageactivity was not due to cleavage by an individual single-chainmeganuclease but, rather, by a intermolecular dimer of two single-chainmeganucleases in which one domain from each associated to form afunctional meganuclease that effectively behaves like the wild-typehomodimer. Indeed, a substantial portion of the N-terminal I-CreIsubunit (amino acids 94-163) was removed in the production of thesingle-chain meganuclease reported by Epinat et al. An inspection of thethree-dimensional I-CreI crystal structure (Jurica et al. (1998), Mol.Cell 2:469-476) reveals that this truncation results in the removal ofthree alpha-helices from the surface of the N-terminal subunit and thesubsequent exposure to solvent of a significant amount of hydrophobicsurface area. As such, the present inventors hypothesized that theN-terminal subunit from the single-chain I-CreI meganuclease of Epinatet al. is unstable and inactive and that the observed DNA cleavageactivity is, in fact, due to the dimerization of the C-terminal subunitsfrom two single-chain proteins. The protein stability problems resultingfrom application of the method of Epinat et al. are also discussed inFajardo-Sanchez et al. (2008). Nucleic Acids Res. 36:2163-2173.

2. Design of Single-Chain LAM Meganucleases Using the Method of Epinatet al.

To more critically evaluate the method for single-chain I-CreImeganuclease production reported by Epinat et al. (Epinat et al. (2003),Nucleic Acids Res. 31: 2952-62; WO 2003/078619), a single-chainmeganuclease was produced in which the N- and C-terminal I-CreI domainsrecognize different DNA half-sites. The method reported in Epinat et al.was used to produce a pair of single-chain meganucleases comprising oneLAM1 domain and one LAM2 domain. This “LAM1epLAM2” meganuclease (SEQ IDNO: 48) comprises an N-terminal LAM domain and a C-terminal LAM2 domainwhile “LAM2epLAM1” (SEQ ID NO: 49) comprises an N-terminal LAM2 domainand a C-terminal LAM domain. In total, both single-chain meganucleasesdiffer by 11 amino acids from that reported by Epinat et al. and allamino acid changes are in regions of the enzyme responsible for DNArecognition which are not expected to affect subunit interaction.

3. Construction of Single-Chain Meganucleases.

LAM1epLAM2 and LAM2epLAM1 were produced by PCR of existing LAM1 and LAM2genes with primers that introduce the I-DmoI linker sequence (whichtranslates to MLERIRLFNMR (SEQ ID NO: 104)) as well as restrictionenzyme sites for cloning. The two LAM subunits were cloned sequentiallyinto pET-21a vectors with a six histidine tag (SEQ ID NO: 110) fused atthe 3′ end of the full-length single-chain gene for purification(Novagen Corp., San Diego, Calif.). All nucleic acid sequences wereconfirmed using Sanger Dideoxynucleotide sequencing (see, Sanger et al.(1977), Proc. Natl. Acad. Sci. 1USA. 74(12): 5463-7).

The LAMep meganucleases were expressed and purified using the followingmethod. The constructs cloned into a pET21a vector were transformed intochemically competent BL21 (DE3) pLysS, and plated on standard 2xYTplates containing 200 j±g/ml carbanicillin. Following overnight growth,transformed bacterial colonies were scraped from the plates and used toinoculate 50 ml of 2XYT broth. Cells were grown at 37° C. with shakinguntil they reached an optical density of 0.9 at a wavelength of 600 nm.The growth temperature was then reduced from 37° C. to 22° C. Proteinexpression was induced by the addition of 1 mM IPTG, and the cells wereincubated with agitation for two and a half hours. Cells were thenpelleted by centrifugation for 10 min. at 6000×g. Pellets wereresuspended in 1 ml binding buffer (20 mM Tris-HCL. pH 8.0, 500 mM NaCl,10 mM imidazole) by vortexing. The cells were then disrupted with 12pulses of sonication at 50% power and the cell debris was pelleted bycentrifugation for 15 min at 14,000×g. Cell supernatants were diluted in4 ml binding buffer and loaded onto a 200 μl nickel-chargedmetal-chelating Sepharose column (Pharmacia).

The column was subsequently washed with 4 ml wash buffer (20 mMTris-HCl, pH 8.0, 500 mM NaCl, 60 mM imidazole) and with 0.2 ml elutionbuffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 400 mM imidazole).Meganuclease enzymes were eluted with an additional 0.6 ml of elutionbuffer and concentrated to 50-130 μl using Vivospin disposableconcentrators (ISC, Inc., Kaysville, Utah). The enzymes were exchangedinto SA buffer (25 mM Tris-HCL, pH 8.0, 100 mM NaCl, 5 mM MgCl₂, 5 mMEDTA) for assays and storage using Zeba spin desalting columns (PierceBiotechnology, Inc., Rockford, Ill.). The enzyme concentration wasdetermined by absorbance at 280 nm using an extinction coefficient of23,590 M⁻¹cm⁻¹. Purity and molecular weight of the enzymes was thenconfirmed by MALDI-TOF mass spectrometry.

4. Cleavage Assays.

All enzymes purified as described above were assayed for activity byincubation with linear, double-stranded DNA substrates containingmeganuclease recognition sequences. Synthetic oligonucleotidescorresponding to both sense and antisense strands of the recognitionsequences were annealed and were cloned into the SmaI site of the pUC19plasmid by blunt-end ligation. The sequences of the cloned binding siteswere confirmed by Sanger dideoxynucleotide sequencing. All plasmidsubstrates were linearized with XmnI or ScaI concurrently with themeganuclease digest. The enzyme digests contained 5 μl 0.05 μM DNAsubstrate, 2.5 μl 5 μM single-chain meganuclease, 9.5 μl SA buffer, and0.5 μl XmnI or ScaI. Digests were incubated at either 37° C. for fourhours. Digests were stopped by adding 0.3 mg/ml Proteinase K and 0.5%SDS, and incubated for one hour at 37° C. Digests were analyzed on 1.5%agarose and visualized by ethidium bromide staining.

5. Results

The LAMep meganucleases produced using the method of Epinat et al. wereincubated with DNA substrates comprising the LAM1 palindrome (SEQ IDNOs: 40 and 41), the LAM2 palindrome (SEQ ID NOs. 44 and 45), or theLAM1/LAM2 hybrid site (SEQ ID NOs. 46 and 47). The LAM1epLAM2single-chain meganuclease was found to cleave primarily the LAM2palindrome whereas the LAM2epLAM1 single-chain meganuclease was found tocleave primarily the LAM1 palindrome. Neither single-chain meganucleasecleaved the hybrid site to a significant degree. These results suggestthat, indeed, the method of Epinat et al. produces single-chainmeganucleases that are unable to cleave non-palindromic DNA sequences.Both single-chain meganucleases were found to cleave primarily therecognition sequence corresponding to a palindrome of the half-siterecognized by the C-terminal subunit, suggesting that the N-terminalsubunit is inactive. Thus, the active meganuclease species characterizedby Epinat et al. appears to be primarily a dimer between the C-terminalsubunits of a pair of single-chain I-CreI meganucleases. Alternatively,cleavage of the palindromic DNA site may be due to sequential singlestrand nicking by the C-terminal subunits of different single-chainI-CreI meganucleases. In either case, in contrast to claims made byEpinat et al., the method does not produce a substantially functionalsingle-chain I-CreI heterodimer and is generally not useful for therecognition and cleavage of non-palindromic DNA sites.

Example 2 Single-Chain I-CreI Meganucleases Produced Using a FlexibleGly-Ser Linker 1. Design of Single-Chain LAM Meganucleases Using aGly-Ser Linker

The designed LAM1 and LAM2 endonucleases were fused into a singlepolypeptide using Linker 3 from Table 3. Val-151 was used as theN-terminal fusion point (to the LAM subunit) while Phe-9 was theC-terminal fusion point (to the LAM2 subunit). The resultingsingle-chain meganuclease, “LAM1gsLAM2” (SEQ ID NO: 50) was cloned intopET21a, expressed in E. coli and purified as described in Example 1.

2. Results

LAM1gsLAM2 was assayed for cleavage activity using the same DNAsubstrates and incubation conditions as described in Example 1. Incontrast to results with the LAMep meganucleases, LAM1gsLAM2 was foundto cleave primarily the hybrid LAM1/LAM2 recognition sequence (SEQ IDNOs: 46 and 47). The extent of cleavage is significantly reducedrelative to the LAM1/LAM2 heterodimer produced by co-expressing the LAM1and LAM2 monomers in E. coli. Under the same reaction conditions, theheterodimer cleaves the LAM1/LAM2 recognition sequence to completion,suggesting that the Gly-Ser linker impairs cleavage activity to someextent. Nonetheless, LAM1gsLAM2 exhibits a much stronger preference forthe hybrid site over the palindromic LAM1 or LAM2 sites and, so hasutility for applications in which specificity is of greater importancethan activity.

Example 3 Single-Chain I-CreI Meganucleases Produced Using a StructuredLinker 1. Design of Single-Chain LAM Meganucleases Using a Designed,Structured Linker

The designed LAM1 and LAM2 endonucleases were fused into a singlepolypeptide using Linker 9 from Table 6. Asp-153 was used as theN-terminal fusion point (to the LAM1 subunit) while Lys-7 was theC-terminal fusion point (to the LAM2 subunit). The resultingsingle-chain meganuclease, “LAM1desLAM2” (SEQ ID NO: 51) was cloned intopET21a, expressed in E. coli and purified as described in Example 1.

2. Results

LAM1desLAM2 was assayed for cleavage activity using the same DNAsubstrates and incubation conditions as described in Example 1. Incontrast to results with the LAMep meganucleases, LAM1desLAM2 was foundto cleave primarily the hybrid LAM1/LAM2 recognition sequence (SEQ IDNO: 46 and 47). The extent of cleavage is comparable to the LAM1/LAM2heterodimer produced by co-expressing the LAM1 and LAM2 monomers in E.coli. These results suggest that designed, structured linkers such asLinker 9 do not interfere significantly with cleavage activity.Moreover, LAM1desLAM2 is structurally stable and maintains catalyticactivity for >3 weeks when stored in SA buffer at 4° C. Importantly,LAM1desLAM2 exhibits minimal activity toward the palindromic LAM1 andLAM2 sites (SEQ ID NOS: 40 and 41 and 44 and 45), indicating that thefunctional species produced by the method disclosed here is primarily asingle-chain heterodimer.

Example 4 Single-Chain I-MsoI Meganucleases Produced Using a StructuredLinker 1. Design of Single-Chain I-MsoI Meganucleases Using a Designed,Structured Linker

A pair of I-MsoI endonuclease subunits (unmodified with respect to DNAcleavage specificity) were fused into a single polypeptide using Linker30 from Table 8. Ile-166 was used as the N-terminal fusion point whileLeu-7 was the C-terminal fusion point. The resulting single-chainmeganuclease, “MSOdesMSO” (SEQ ID NO: 52) was cloned into pET21a with aC-terminal 6×His-tag (SEQ ID NO: 110) to facilitate purification. Themeganuclease was then expressed in E. coli and purified as described inExample 1.

2. Results

Purified MSOdesMSO was assayed for the ability to cleave a plasmidsubstrate harboring the wild-type I-MsoI recognition sequence (SEQ IDNO:53 and SEQ ID NO:54 and 54) under the incubation conditions asdescribed in Example 1. The enzyme was found to have cleavage activitycomparable to the I-MsoI homodimer (which, in this case, is expected torecognize and cut the same recognition sequence as MSOdesMSO). SDS-PAGEanalyses revealed that MSOdesMSO has an apparent molecular weight of ˜40kilodaltons, consistent with it being a pair of covalently joined I-MsoIsubunits, and no protein degradation products were apparent. Theseresults indicate that the invention is suitable for the production ofstable, high-activity single-chain meganucleases derived from I-MsoI.

TABLE 11 I-CreI Modifications from WO 2007/047859 Favored Sense-StrandBase Posn. A C G T A/T A/C A/G C/T G/T A/G/T A/C/G/T -1 Y75 R70* K70Q70* T46* G70 L75* H75* E70* C70 A70 C75* R75* E75* L70 S70 Y139* H46*E46* Y75* G46* C46* K46* D46* Q75* A46* R46* H75* H139 Q46* H46* -2 Q70E70 H70 Q44* C44* T44* D70 D44* A44* K44* E44* V44* R44* I44* L44* N44*-3 Q68 E68 R68 M68 H68 Y68 K68 C24* F68 C68 I24* K24* L68 R24* F68 -4A26* E77 R77 S77 S26* Q77 K26* E26* Q26* -5 E42 R42 K28* C28* M66 Q42K66 -6 Q40 E40 R40 C40 A40 S40 C28* R28* I40 A79 S28* V40 A28* C79 H28*I79 V79 Q28* -7 N30* E38 K38 I38 C38 H38 Q38 K30* R38 L38 N38 R30* E30*Q30* Favored Sense-Strand Base Posn. A Posn. A Posn. A Posn. A Posn. APosn. A -8 F33 E33 F33 L33 R32* R33 Y33 D33 H33 V33 I33 F33 C33 -9 E32R32 L32 D32 S32 K32 V32 I32 N32 A32 H32 C32 Q32 T32 Bold entries arewild-type contact residues and do not constitute “modifications” as usedherein. An asterisk indicates that the residue contacts the base on theantisense strand.

TABLE 12 I-MsoI Modifications from WO 2007/047859 Favored Sense-StrandBase Position A C G T −1 K75* D77 K77 C77 Q77 E77 R77 L77 A49* K49* E49*Q79* C49* R75* E79* K79* K75* R79* K79* −2 Q75 E75 K75 A75 K81 D75 E47*C75 C47* R47* E81* V75 I47* K47* I75 L47* K81* T75 R81* Q47* Q81* −3 Q72E72 R72 K72 C26* Y72 K72 Y72 L26* H26* Y26* H26* V26* K26* F26* A26*R26* I26* −4 K28 K28* R83 K28 Q83 R28* K83 K83 E83 Q28* −5 K28 K28* R45Q28* C28* R28* E28* L28* I28* −6 I30* E43 R43 K43 V30* E85 K43 I85 S30*K30* K85 V85 L30* R30* R85 L85 Q43 E30* Q30* D30* −7 Q41 E32 R32 K32 E41R41 M41 K41 L41 I41 −8 Y35 E32 R32 K32 K35 K32 K35 K35 R35 −9 N34 D34K34 S34 H34 E34 R34 C34 S34 H34 V34 T34 A34 Bold entries are representwild-type contact residues and do not constitute “modifications” as usedherein. An asterisk indicates that the residue contacts the base on theantisense strand.

TABLE 13 I-Ceu Modifications from WO 2007/047859 Favored Sense-StrandBase Position A C G T −1 C92* K116* E116* Q116* A92* R116* E92* Q92*V92* D116* K92* −2 Q117 E117 K117 C117 C90* D117 R124 V117 L90* R174*K124 T117 V90* K124* E124* Q90* K90* E90* R90* D90* K68* −3 C70* K70*E70* Q70* V70* E88* T70* L70* K70* −4 Q126 E126 R126 K126 N126 D126 K126L126 K88* R88* E88* Q88* L88* K88* D88* C88* K72* C72* L72* V72* −5 C74*K74* E74* C128 L74* K128 L128 V74* R128 V128 T74* E128 T128 −6 Q86 D86K128 K86 E86 R128 C86 R84* R86 L86 K84* K86 E84* −7 L76* R76* E76* H76*C76* K76* R84 Q76* K76* H76* −8 Y79 D79 R79 C79 R79 E79 K79 L79 Q76 D76K76 V79 E76 R76 L76 −9 Q78 D78 R78 K78 N78 E78 K78 V78 H78 H78 L78 K78C78 T78 Bold entries are wild-type contact residues and do notconstitute “modifications” as used herein. An asterisk indicates thatthe residue contacts the base on the antisense strand.

TABLE 14 I-SceI Modifications from WO 2007/047859 Favored Sense-StrandBase Position A C G T 4 K50 R50* E50* K57 K50* R57 M57 E57 K57 Q50* 5K48 R48* E48* Q48* Q102 K48* K102 C102 E102 R102 L102 E59 V102 6 K59R59* K84 Q59* K59* E59* Y46 7 C46* R46* K86 K68 L46* K46* R86 C86 V46*E86 E46* L86 Q46* 8 K61* E88 E61* K88 S61* R61* R88 Q61* V61* H61* K88H61* A61* L61* 9 T98* R98* E98* Q98* C98* K98* D98* V98* L98* 10 V96*K96* D96* Q96* C96* R96* E96* A96* 11 C90* K90* E90* Q90* L90* R90* 12Q193 E165 K165 C165 E193 R165 L165 D193 C193 V193 A193 T193 S193 13C193* K193* E193* Q193* L193* R193* D193* C163 D192 K163 L163 R192 14L192* E161 K147 K161 C192* R192* K161 Q192* K192* R161 R197 D192* E192*15 E151 K151 C151 L151 K151 17 N152* K152* N152* Q152* S152* K150* S152*Q150* C150* D152* L150* D150* V150* E150* T150* 18 K155* R155* E155*H155* C155* K155* Y155* Bold entries are wild-type contact residues anddo not constitute “modifications” as used herein. An asterisk indicatesthat the residue contacts the base on the antisense strand.

1-9. (canceled)
 10. A recombinant single-chain meganuclease comprising:a first LAGLIDADG subunit derived from a first mono-LAGLIDADGmeganuclease, said first LAGLIDADG subunit having a first recognitionhalf-site; a second LAGLIDADG subunit derived from a secondmono-LAGLIDADG meganuclease or a di-LAGLIDADG meganuclease, said secondLAGLIDADG subunit having a second recognition half-site; wherein saidfirst and second LAGLIDADG subunits are covalently joined by apolypeptide linker such that said first LAGLIDADG domain is N-terminalto said linker and said second LAGLIDADG domain is C-terminal to saidlinker; and wherein said first and second LAGLIDADG subunits are capableof functioning together to recognize and cleave a non-palindromic DNAsequence which is a hybrid of said first recognition half-site and saidsecond recognition half-site.
 11. The recombinant single-chainmeganuclease of claim 10 wherein: the first LAGLIDADG subunit is derivedfrom a mono-LAGLIDADG meganuclease selected from the group consisting ofI-Crel, I-Msol and I-Ceul; and the second LAGLIDADG subunit is derivedfrom either (1) a mono-LAGLIDADG meganuclease selected from the groupconsisting of I-Crel, I-Msol and I-Ceul, or (2) a di-LAGLIDADGmeganuclease selected from the group consisting of I-Dmol, I-Scel andI-Anil.
 12. The recombinant single-chain meganuclease of claim 10wherein: the first LAGLIDADG subunit is derived from a different speciesthan the second LAGLIDADG subunit.
 13. The recombinant single-chainmeganuclease of claim 10 wherein: said first LAGLIDADG subunit comprisesa polypeptide sequence having at least 85% sequence identity to a firstLAGLIDADG domain selected from the group consisting of residues 9-151 ofa wild-type I-Crel meganuclease of SEQ ID NO: 1; residues 11-162 of awild-type I-Msol meganuclease of SEQ ID NO: 2; and residues 55-210 of awild-type I-Ceul meganuclease of SEQ ID NO:
 3. 14. The recombinantsingle-chain meganuclease of claim 11 wherein: said second LAGLIDADGsubunit comprises a polypeptide sequence having at least 85% sequenceidentity to a second LAGLIDADG domain selected from the group consistingof residues 9-151 of a wild-type I-Crel meganuclease of SEQ ID NO: 1;residues 11-162 of a wild-type I-Msol meganuclease of SEQ ID NO: 2;residues 55-210 of a wild-type I-Ceul meganuclease of SEQ ID NO: 3;residues 9-96 of a wild-type I-Dmol of SEQ ID NO: 4; residues 105-178 ofa wild-type I-Dmol of SEQ ID NO: 4; residues 32-123 of a wild-typeI-Scel of SEQ ID NO: 5; residues 134-225 of a wild-type I-Scel of SEQ IDNO: 5; residues 4-121 of a wild-type I-Anil of SEQ ID NO: 6; andresidues 136-254 of a wild-type I-Anil of SEQ ID NO:
 6. 15. Therecombinant single-chain meganuclease of claim 11 wherein: each of saidLAGLIDADG subunits comprises at least 85% identity to a LAGLIDADG domainindependently selected from the group consisting of residues 9-151 of awild-type I-Crel meganuclease of SEQ ID NO: 1; residues 11-162 of awild-type I-Msol meganuclease of SEQ ID NO: 2; residues 55-210 of awild-type I-Ceul meganuclease of SEQ ID NO: 3; residues 9-96 of awild-type I-Dmol of SEQ ID NO: 4; residues 105-178 of a wild-type I-Dmolof SEQ ID NO: 4; residues 32-123 of a wild-type I-Scel of SEQ ID NO: 5;residues 134-225 of a wild-type I-Scel of SEQ ID NO: 5; residues 4-121of a wild-type I-Anil of SEQ ID NO: 6; and residues 136-254 of awild-type I-Anil of SEQ ID NO: 6; and at least one of said LAGLIDADGdomains comprises at least one amino acid modification disclosed in anyof Tables 11, 12, 13 and
 14. 16. The recombinant single-chainmeganuclease of claim 15 wherein: at least one LAGLIDADG domain isderived from I-Crel and at least one modification is selected from Table1 of any of Tables 11, 12, 13 and 14; at least one LAGLIDADG domain isderived from I-Msol and at least one modification is selected from Table12; at least one LAGLIDADG domain is derived from I-Ceul and at leastone modification is selected from Table 13; or at least one LAGLIDADGdomain is derived from I-Scel and at least one modification is selectedfrom Table
 14. 17. The recombinant single-chain meganuclease of claim 11wherein: each of said LAGLIDADG subunits has a recognition half-siteselected from the group consisting of SEQ ID NOs: 7-30.
 18. Therecombinant single-chain meganuclease of claim 17 wherein: at least oneof said LAGLIDADG subunits has a recognition half-site selected from thegroup consisting of SEQ ID NOs: 7-30; and the other of said LAGLIDADGsubunits has a recognition half-site which differs by at modification ofat least one base pair from a recognition half-site selected from thegroup consisting of SEQ ID NOs: 7-30.
 19. The recombinant single-chainmeganuclease of claim 10 wherein: said polypeptide linker is a flexiblelinker.
 20. The recombinant single-chain meganuclease of claim 19wherein: said linker comprises 15-40 residues.
 21. The recombinantsingle-chain meganuclease of claim 19 wherein: said linker comprises25-31 residues.
 22. The recombinant single-chain meganuclease of claim19 wherein: at least 50% of said linker comprises polar unchargedresidues.
 23. The recombinant single-chain meganuclease of claim 10wherein: said polypeptide linker has a stable secondary structure. 24.The recombinant single-chain meganuclease of claim 23 wherein: saidstable secondary structure comprises at least two α-helix structures.25. The recombinant single-chain meganuclease of claim 23 wherein: saidstable secondary structure comprises from N-terminus to C-terminus afirst loop, a first α-helix, a first turn, a second α-helix, and asecond loop.
 26. The recombinant single-chain meganuclease of claim 23wherein: said linker comprises 23-56 residues.